Therapeutic and diagnostic conjugates for use with multispecific antibodies

ABSTRACT

Disclosed are compounds that include two or more haptens conjugated by a spacer or a carrier. The haptens may include diethylenetriaminepentaacetate (DTPA), histimine-succinyl-glutamine (HSG), or combinations of DTPA and HSG. The compound also includes an effector molecule which may be conjugated to one or more of the haptens, the spacer/carrier, or both. The effector molecule may be conjugated by a number of linkages including an ester linkage, an imino linkage, an amino linkage, a sulfide linkage, a thiosemicarbazone linkage, a semicarbazone linkage, an oxime linkage, an ether linkage, or combinations of these linkages. Also disclosed are methods of synthesizing the compounds and/or precursors of the compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.10/150,654, filed May 17, 2002; which is a continuation-in-part of U.S.application Ser. No. 09/382,186, filed Aug. 23, 1999 and acontinuation-in-part of U.S. application Ser. No. 09/823,746, filed Apr.3, 2001; both of which are continuations-in-part of U.S. applicationSer. No. 09/337,756, filed Jun. 22, 1999; which claims the benefit under35 U.S.C.§119(e) to U.S. Application No. 60/090,142, filed Jun. 22,1998, and to U.S. Application No. 60/104,156, filed Oct. 14, 1998. Thecontents of all the aforementioned applications are incorporated hereinby reference in their entireties.

BACKGROUND

A general approach to cancer therapy and diagnosis involves directingantibodies or antibody fragments to disease tissues, whereby theantibody or antibody fragment can target a diagnostic agent ortherapeutic agent to the disease site. One specific approach to thismethodology which has been under investigation, involves the use ofbsAbs having at least one arm that specifically binds a targeteddiseased tissue and at least one other arm that specifically binds a lowmolecular weight hapten. In this methodology, a bsAb is administered andallowed to localize to a target and to clear normal tissue. Some timelater, a radiolabeled low molecular weight hapten is given, which, beingrecognized by the second specificity of the bsAb, also localizes to theoriginal target.

Although low MW haptens used in combination with bsAbs possess a largenumber of specific imaging and therapy uses, it is impractical toprepare individual bsAbs for each possible application. Further, theapplication of a bsAb/low MW hapten system has several otherrequirements. First, the arm of the bsAb that binds to the low MW haptenmust bind with high affinity, because a low MW hapten is designed toclear the living system rapidly when not bound by bsAb. Second, thenon-bsAb-bound low MW hapten actually needs to clear the living systemrapidly to avoid non-target tissue uptake and retention. Third, thedetection and/or therapy agent must remain associated with the low MWhapten throughout its application within the bsAb protocol.

Of interest with this approach are bsAbs that direct chelators and metalchelate complexes to cancers using Abs of appropriate dual specificity.The chelators and metal chelate complexes used are often radioactive,using radionuclides for radioimmuno-imaging. (See Goodwin et al., U.S.Pat. No. 4,863,713 (describing the use of cobalt-57); Barbet et al.,U.S. Pat. No. 5,256,395 and U.S. Pat. No. 5,274,076; Goodwin et al., J.Nucl. Med., 33:1366-1372 (1992); and Kranenborg et al., Cancer Res(suppl.), 55:5864s-5867s (1995) and Cancer (suppl.) 80:2390-2397 (1997)(all describing the use of indium-111); and Boden et al., BioconjugateChem., 6:373-379, (1995); and Schuhmacher et al., Cancer Res.,55:115-123 (1995)(describing the use of gallium-68)). Because the Absare raised against the chelators and metal chelate complexes, they haveremarkable specificity for the complex against which they wereoriginally raised. Indeed, the bsAbs of Boden et al. have specificityfor single enantiomers of enantiomeric mixtures of chelators andmetal-chelate complexes. This great specificity has proven to be adisadvantage in one respect, in that other nuclides (such as yttrium-90and bismuth-213 useful for radioimmunotherapy (RAIT), and gadoliniumuseful for MRI), cannot be readily substituted into available reagentsfor alternative uses. As a result iodine-131, a non-metal, has beenadopted for RAIT purposes by using an I-131-labeled indium-metal-chelatecomplex in the second targeting step. Another disadvantage to thismethodology requires that antibodies be raised against every agentdesired for diagnostic or therapeutic use.

As such, pretargeting methodologies have received considerable attentionfor cancer imaging and therapy. Unlike direct targeting systems where aneffector molecule (e.g., a radionuclide or a drug linked to a smallcarrier) is directly linked to the targeting agent (e.g., a bindingmolecule such as a bsAb), in pretargeting systems, the effector moleculeis given some time after the targeting agent. This allows time for thetargeting agent to localize in tumor lesions and, more importantly,clear from the body. Because most targeting agents have been bindingproteins such as antibodies, they tend to clear much more slowly fromthe body (usually days) than the smaller effector molecules (usually inminutes). As such, in direct targeting systems involving therapeuticradionuclides, the body, and in particular the highly vulnerable redmarrow, may be exposed to the radiation all the while the targetingagent is slowly reaching its peak levels in the tumor and clearing fromthe body. However, in a pretargeting system, the radionuclide (i.e., aneffector) is usually bound to a small “carrier” molecule, such as achelate or peptide, which clears very quickly from the body, and thusexposure of normal tissues is minimized. In a pretargeting system,maximum tumor uptake of the radionuclide is also very rapid because thesmall carrier molecule efficiently transverses the tumor vasculature andbinds to the primary targeting agent. The small size of a carriermolecule may also encourage a more uniform distribution in the tumor.

Pretargeting methods have used a number of different strategies, butoften involve an avidin/streptavidin-biotin recognition system orbi-specific antibodies that co-recognize a tumor antigen and one or molehaptens on the carrier molecule, which includes an effector molecule.The avidin/streptavidin system is highly versatile and has been used inseveral configurations. In this system, antibodies coupled withstreptavidin or biotin are used as the primary targeting agent. This isfollowed sometime later by administration of the effector molecule,which may be conjugated with biotin or with avidin/streptavidin,respectively. Another configuration relies on a 3-step approach: (1)first targeting a biotin-conjugated antibody; (2) followed by a bridgingwith streptavidin/avidin; and (3) then the biotin-conjugated effector isgiven. These systems can be easily converted for use with a variety ofeffector substances so long as the effector and the targeting agent canbe coupled with biotin or streptavidin/avidin depending on theconfiguration used. With its versatility for use in many targetingsituations and high binding affinity between avidin/streptavidin andbiotin, this type of pretargeting has considerable advantages over otherproposed systems. However, avidin and streptavidin are foreign proteinsand therefore can be immunogenic, which may limit the number of timesthey can be administered in a clinical application. In this respect,bsAbs have the advantage of being able to be engineered as a relativelynon-immunogenic humanized protein. Although the binding affinity of absAb (typically 10⁻⁹ to 10⁻¹⁰ M) cannot compete with the extremely highaffinity of the streptavidin/avidin-biotin affinity (˜10⁻¹⁵ M), bothpretargeting systems are dependent on the binding affinity of theprimary targeting agent, and therefore the higher affinity of thestreptavidin/avidin-biotin systems may not offer a substantial advantageover a bsAb pretargeting system. However, most bsAbs have only one armavailable for binding the primary target, whereas thestreptavidin/avidin-biotin pretargeting systems typically use a wholeIgG with two arms for binding the target, which strengthens targetbinding. By using a divalent peptide, an affinity enhancement may beachieved, which can greatly improve the binding of the peptide to thetarget site compared to a monovalent peptide. Thus, both systems canprovide excellent targeting ratios with reasonable retention.

Pretargeting with a bsAb also requires one arm of the antibody torecognize an effector molecule or a molecule that contains an effectormolecule (e.g., a carrier with an effector together as a “targetableconstruct”). Most radionuclide targeting systems reported to date haverelied on an antibody to a chelate-metal complex, such as antibodiesdirected against indium-loaded DTPA or antibodies to other chelates.Because the antibody is generally selective for a particularchelate-metal complex, new bsAbs typically need to be constructed foreach selected chelate-metal complex. This can be avoided by using acarrier molecule that includes the effector molecule and a hapten, whichis specifically recognized by the antibody. As such, the carrier,including the effector and hapten, functions as a targetable construct.The targetable construct is “modular” in nature, in that differenteffectors can be included in the construct without having to use adifferent antibody in the pretargeting system, because the antibodyrecognizes the hapten on the targetable construct. In this way, avariety of effectors can be used in the pretargeting system, providedthat the targetable construct that includes the effector maintains thesame recognized hapten.

Because in a pre-targeting method the effector molecule (i.e., targetingmolecule or carrier molecule) and the binding molecule (i.e., thetargeting construct or antibody) are not administered concurrently, thebinding molecule must not be internalized by the targeted tissue priorto administering the effector molecule. However, because the bindingmolecule is bivalent and bispecific, internalization of the bindingmolecule may be hindered or delayed until after the effector molecule isadministered, even if the binding molecule recognizes an antigen that ispart of an internalizing receptor on the surface of the targeted tissue.Further, if the effector molecule is multivalent (i.e., it has two ormore moieties recognized by the binding molecule), the effector moleculecan crosslink two or more binding molecules on the surface of thetargeted tissue to facilitate internalization of the crosslinkedcomplex. The effector molecule may also include one or more moietiesthat facilitate internalization by binding to internalizing receptors onthe surface of the targeted tissue (e.g., the folate receptor). Methodsof compositions for administering therapeutic and diagnostic agents aredescribed in U.S. 60/444,357, filed Jan. 31, 2003.

Thus, there is a continuing need for immunological agents which can bedirected to diseased tissue and can specifically bind to a subsequentlyadministered targetable diagnostic or therapeutic conjugate, and aflexible, modular system that accommodates different diagnostic andtherapeutic agents without alteration to the bi-specific ormulti-specific antibodies. We have continued to develop the pretargetingsystem originally described by Janevik-Ivanovska et al. that used anantibody directed against a histamine derivative,histamine-succinyl-glycl (HSG), as the recognition system on which avariety of effector substances could be prepared. Excellent pretargetingresults have been reported using a radioiodinated and a rhenium-labeleddivalent HSG-containing peptide. In the present work, we have expandedthis system to include peptides that include haptens and/or chelatorssuch as DTPA, and which may be suitable for radiolabeling with ⁹⁰Y,¹¹¹In, and ¹⁷⁷Lu, as well as ^(99m)Tc.

SUMMARY

Disclosed herein are reagents for therapeutic use, for example, inradioimmunotherapy (RAIT), and diagnostic use, for example, inradioimmunodetection (RAID) and magnetic resonance imaging (MRI). Inparticular, disclosed herein are targetable molecules for use withbinding molecules (i.e. targeting molecules), such as bi-specificantibodies (bsAb) and bi-specific antibody fragments (bsFab) that haveat least one arm that specifically binds the targetable construct and atleast one other arm that specifically binds a targeted tissue.

The compounds described herein include two or more haptens conjugated bya spacer. The haptens may include diethylenetriaminepentaacetate (DTPA),histimine-succinyl-glutamine (HSG), or combinations of DTPA and HSG.Preferably, the compound includes DTPA. In one embodiment, the compoundincludes DTPA and HSG. The compounds may be multivalent to facilitatecrosslinking of one or more binding molecules on the surface of atargeted tissue to facilitate internalization of the crosslinkedcomplex. The compounds may also include one or more moieties thatfacilitate internalization by binding to an internalized receptor on thesurface of the targeted tissue (e.g., the folate receptor).

The compound also includes an effector molecule which may be conjugatedto one or more of the haptens, the spacer, or both. As such, the haptensand/or the spacer may function as carrier molecules for the effector.The effector molecule may be conjugated by a number of linkages, andpreferably, the linkage is stable under physiological conditions inserum, but the linkage is sensitive to hydrolysis when the compounds arelocalized to target cells or internalized by target cells. For example,the linkages may be subject to acid hydrolysis under the physiologicalconditions present within lysosomes. Alternatively, hydrolysis of aparticular linkage may be catalyzed by one or more enzymes localized atthe target cells or internal to the target cells. Suitable linkages mayinclude an ester linkage, an imino linkage, an amino linkage, a sulfidelinkage, a thiosemicarbazone linkage, a semicarbazone linkage, an oximelinkage, an ether linkage, or combinations of these linkages.

The compound may also include metal ions. Preferably, the compoundincludes indium cations. In one embodiment, metal ions, such as indium,are chelated by a hapten such as DTPA.

The spacer may include one or more amino acids, and preferably thespacer includes three or more amino acids. In one embodiment, thepeptide may include one or more D-amino acids, (e.g., to create a morestable molecule that is not easily metabolized in serum).

In one particular embodiment the spacer includes a peptide with one ormore lysine residues and one or more cysteine residues. In anotherembodiment, the spacer includes a penicillamine moiety or a moiety thatis a derivative of penicillamine. In a further embodiment, the spacerincludes a thiolactic acid moiety or a moiety that is a derivative ofthiolactic acid.

The haptens and/or effectors may be conjugated to one or more residuesof the spacer. For example, the haptens may be conjugated to an6-nitrogen atom of a lysine residue, or a sulfur atom of a cysteineresidue. In another example, the effector is conjugated to apenicillamine moiety or a derivative thereof, or a thiolactic acidmoiety or a derivative thereof. Preferably, the effector molecule islinked by an ester linkage, or another linkage which may be hydrolyzedunder physiological conditions after being administered to a subject.

As used herein, an effector molecule includes any molecule that bringsabout a desirable result. As such, an effector molecule many includedrugs, prodrugs, toxins, enzymes, radioisotopes, immunomodulators,cytokines, hormones, nucleotide sequences (e.g., antisense nucleotidesor interference RNAs), binding molecules (e.g., antibodies), orcombinations of these types of molecules. Examples of antisenseoligonucleotides and interference RNAs are disclosed in Kalota et al.,Cancer Biol. Ther. 2004 Jan; 3(1); Tong et al., Clin. Lung Cancer 2001February; 2(3):220-6; Dean et al., Oncogene 2003 Dec. 8; 22(56):9087-96; Nahta et al., Semin. Oncol. 2003 October; 30(5 Suppl 16):143-9; Patry et al., Cancer Res. 2003 Nov. 15; 63(22): 7679Duxbury etal., Biochem Biophys Res Commun. 2003 Nov. 21; 311(3) 786-92;Crnkovic-Mertens et al., Oncogene 2003 Nov. 13; 22(51): 8330-6; Lipscombet al., Clin Exp Metastasis 2003; 20(6): 569-76; Wall et al., Lancet2003 Oct. 25; 362(9393): 1401-3; Bedford et al., Semin Cancer Biol 2003August; 13(40): 301-8; Damm-Welk et al., Semin Cancer Biol. 2003 August;13(4): 283-92; Duursma et al., Semin Cancer Biol. 2003 August; 13(4):267-73, all of which are incorporated herein by reference in theirentireties.

An effector may also include a lipid or a polymer, which may be capableof forming a higher-ordered structure, (e.g., a micelle, liposome, orpolymeric structure), which may incorporate other effectors as describedherein. Alternatively, the effector may be a higher-ordered structureitself (e.g., a micelle, liposome, polymeric structure, and/or ananoparticle). Where the effector is a lipid, the lipid-conjugatedcompound may form an emulsion that is associated with any of theeffectors as described herein.

Therapeutic effector molecules may include cytotoxic drugs, such asaplidin, azaribine, anastrozole, azacytidine, bleomycin, bortezomib,bryostatin-1, busulfan, calicheamycin, camptothecin,10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin,irinotecan (CPT-11), SN-38, carboplatin, cladribine, cyclophosphamide,cytarabine, dacarbazine, docetaxel, dactinomycin, daunomycinglucuronide, daunorubicin, dexamethasone, diethylstilbestrol,doxorubicin, 2-pyrrolinodoxorubicin (2P-DOX), cyano-morpholinodoxorubicin, doxorubicin glucuronide, epirubicin glucuronide, ethinylestradiol, estramustine, etoposide, etoposide glucuronide, etoposidephosphate, floxuridine (FUdR), 3′,5′—O-dioleoyl-FudR (FUdR-dO),fludarabine, flutamide, fluorouracil, fluoxymesterone, gemcitabine,hydroxyprogesterone caproate, hydroxyurea, idarubicin, ifosfamide,L-asparaginase, leucovorin, lomustine, mechlorethamine,medroprogesterone acetate, megestrol acetate, melphalan, mercaptopurine,6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin,mitotane, phenyl butyrate, prednisone, procarbazine, paclitaxel,pentostatin, PSI-341, semustine streptozocin, tamoxifen, taxanes, taxol,testosterone propionate, thalidomide, thioguanine, thiotepa, teniposide,topotecan, uracil mustard, velcade, vinblastine, vinorelbine,vincristine, ricin, abrin, ribonuclease, onconase, rapLR1, DNase I,Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtheria toxin, Pseudomonas exotoxin, Pseudomonas endotoxin, orcombinations of these.

In one embodiment, the effector molecule may be a prodrug that isactivated after the compound is administered to a subject. For example,a prodrug may be activated after it is localized to a targeted celland/or internalized by the targeted cell. In particular, the prodrug maybe activated by physiological conditions in the cell (e.g., the acidicenvironment of lysosomes). Alternatively, the prodrug may be activatedby one or more enzymes, (e.g., carboxylesterase can activate prodrugssuch as irinotecan (CPT-11). Preferably, the effector molecule includescamptothecin, doxorubicin, or derivatives and/or analogs thereof, andpreferably the effector molecule is conjugated by an ester linkage.Doxorubicin derivatives and/or analogs include 2-pyrrolinodoxorubicin(2P-DOX) and cyano-morpholino doxorubicin.

Where an effector molecule is not water soluble, preferably one or moreof the haptens, the spacer (e.g., a peptide), and/or the linkage makesthe effector molecule more water soluble. In one embodiment, aninsoluble effector molecule may be administered as part of an emulsionor liposome, wherein the lipid that forms the emulsion or liposome maybe conjugated to one or more of the administered compounds (e.g., thetargetable construct). In another embodiment, one or more of thehaptens, the spacer, and/or the linkage may reduce the toxicity of theeffector molecule. In a further embodiment, one or more of the haptens,the spacer, and/or the linkage facilitate localization of the compound(which includes the effector molecule) to a targeted tissue, whilenon-targeted compounds (and/or effector molecules) can be rapidlyexcreted. As such, the biodistribution of the effector molecule may bealtered by conjugating the effector to one or more of the haptens, thespacer, and/or the linkage.

The compound may also include an isotope. Examples include ¹⁸F, ³²P,³³P, ⁴⁵Ti, ⁴⁷Sc, ⁵²Fe, ⁵⁹Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁵Se, ⁷⁷As,⁸⁶Y, ⁸⁹Sr, ⁸⁹Zr, ⁹⁰Y, ⁹⁴Tc, ^(94m)Tc, ⁹⁹Mo, ^(99m)Tc, ¹⁰⁵Pd, ¹⁰⁵Rh,¹¹¹Ag, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁵⁴⁻¹⁵⁸Gd,¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁶⁹Er, ¹⁷⁵Lu, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ¹⁹⁴Ir,¹⁹⁸Au, ¹⁹⁹Au, ²¹¹At, ²¹¹Pb, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²²³Ra, or ²²⁵Ac. Theisotope may be covalently linked to the compound or the isotope may bechelated by a chelating moiety present in the compound (e.g., DTPA).

In particular embodiments, the compound includes a peptide, one or morehaptens, and one or more effector molecules. Further, the peptide mayinclude one or more sequences R¹-Lys(X)—R²-Lys(Y) orLys(X)—R²-Lys(Y)—R¹, where R¹ and R² include one or more amino acids,and where (X) and (Y) include one or more conjugated moieties selectedfrom antigenic molecules, haptens, hard acid chelators, and soft acidchelators. The effector molecule, as described herein, may be conjugatedby a linkage to the haptens and/or one or more amino acids present in R¹or R². Desirably, the linkage is stable in physiological conditions inserum, but the linkage is susceptible to hydrolysis when the compound isinternalized in a cell. For example, the linkage may be susceptible tohydrolysis under the acidic conditions in a lysosome or the linkage maybe susceptible to hydrolysis as facilitated by an enzyme (e.g.,carboxylesterase). Linkages may include an ester linkage, an iminolinkage, an amino linkage, a sulfide linkage, a thiosemicarbazonelinkage, a semicarbazone linkage, an oxime linkage, an ether linkage, anamide, and combinations of these linkages. As noted herein, the effectormolecule may include drugs, prodrugs, toxins, enzymes, radioisotopes,immunomodulators, cytokines, hormones, nucleotide sequences, bindingmolecules, or combinations of these.

The moiety may be a hard acid chelator, and where the compound includesa hard acid chelator, preferably the compound further includes a cationselected from the group consisting of Group IIa and Group IIIa metalcations. The compound may also include one or more isotopes as describedabove.

In one embodiment, the moiety includes DTPA, HSG, DOTA, NOTA, TETA,Tscg-Cys, Tsca-Cys, nitroloacetic acid, or combinations of thesemoieties. Preferably, the compound includes DTPA, HSG, or combinationsof DTPA and HSG. Most preferably, the compound includes DTPA. Themoieties, designated by (X) and (Y), may be the same or different.

The compound may also include a soft acid chelator. Where the compoundincludes a soft acid chelator, the compound may also include a cationselected from the group consisting of transition metals, Bi,lanthanides, and actinides. For example, the compound may include Tc,Re, Bi, or combinations of these cations.

It may be desirable to synthesis peptides that include particular aminoacids or types of amino acids. For example, in one embodiment the groupdesignated by R² may include tyrosine. Also, it may be desirable tocreate a peptide that includes one or more D-amino acids.

Also disclosed herein is a method of treating and/or diagnosing adisease or condition that may lead to a disease in a patient, which mayinclude: (1) administering a binding molecule to the patient, where thebinding molecule has at least one arm that binds a targeted tissue andat least one other arm that binds a targetable construct; (2)optionally, administering a clearing composition to the patient andallowing the composition to clear non-localized binding molecules fromcirculation; and (3) administering to the patient one or more targetableconstructs that include one or more of the above-described compounds.For example, the targetable construct may include one or more compoundsthat include: (1) two or more haptens linked by a spacer, where one ormore haptens are DTPA or HSG; and (2) one or more effector moleculesconjugated to one or more of the haptens, the spacer, or both. In oneembodiment the targetable construct includes a compound that includes:(1) a peptide having one or more of the sequences R¹-Lys(X)—R²-Lys(Y) orLys(X)—R²-Lys(Y)—R¹, where R¹ and R² include one or more amino acids andwhere (X) and (Y) include a conjugated moiety; and (2) an effectormolecule conjugated to the peptide. The moiety may include an antigenicmolecule, a hapten, a hard acid chelator, a soft acid chelator orcombinations of these types of moieties.

As used herein, a binding molecule (i.e., a targeting molecule) mayinclude an antibody or a fragment of an antibody. Particular suitableantibodies or binding molecules may be multivalent and multispecific(e.g., bi-specific antibodies). The binding molecule may include amonoclonal antibody or a fragment of a monoclonal antibody. The antibodyor antibody fragment (e.g., monoclonal) may include a human, chimeric orhumanized antibody or a fragment of a human, chimeric or humanizedantibody. Examples of particular suitable antibodies include MAb 679,MAb 734, MAb Mu-9, MN-14, RS-7, 679, 734, or combinations of theseantibodies. The binding molecule or antibody may include a fusionprotein. In some embodiments, it may be desirable to use antibodies,fragments thereof, or binding molecules that include the CDRs of Mab679, Mab 734, Mab Mu-9, MN-14, RS-7, 679, or 734.

As noted herein, the targetable construct may include a peptideincluding the sequence R¹-Lys(X)—R²-Lys(Y) or Lys(X)—R²-Lys(Y)—R¹, andan effector molecule conjugated to an amino acid present in R¹ or R²and/or to one or more of the conjugated moieties (X) and/or (Y).Preferably the effector molecule is conjugated by an ester linkage, anamido linkage, and/or a hydrazone linkage.

Also, as noted herein, the effector molecule may include any moleculethat brings about a desirable result. For example, the effector moleculemay include one or more drugs, prodrugs, toxins, enzymes, radioisotopes,immunomodulators, cytokines, hormones, nucleotide sequences (e.g.,antisense oligonucleotide or interference RNAs), binding molecules, ormolecules that facilitate administration of the foregoing categories ofmolecules (e.g., a lipid or polymer capable of forming a higher-orderedstructure, or a higher-ordered structure itself, such as a micelle,liposome, polymeric structure, and/or nanoparticle), which may be usefulas drug carriers. Specific examples of effector molecules areexemplified herein. In particular, the effector molecule may includecamptothecin or a derivative of camptothecin, (e.g., SN-38,10-hydroxy-CPT, 9-amino-CPT, irinotecan (CPT-11), etc.). Doxorubicin, orderivatives and/or analogs thereof, may also be a particularly suitableeffector molecule. Doxorubicin derivatives are described in Nagy et al.,Proc. Natl. Acad. Sci. USA, 1996, 93:2464-9. Antitumor anthracyclinesmay also be particularly suitable effector molecules, as described inMonneret, Eur. J. Med. Chem. 2001 36:483-93. The effector molecule,(e.g., camptothecin and/or doxorubicin), may be conjugated to thetargetable construct and/or associated with a drug-carrier such as amicelle/liposome or an emulsion, wherein the drug-carrier is conjugatedto the targetable construct.

In regard to selected enzymes as effector molecules, particularlysuitable enzymes may include carboxylesterases, glucuronidases,carboxypeptidases, beta-lactamases, phosphatases, or mixtures of theseenzymes.

The methods of treating and/or diagnosing diseases or conditions may beused to treat/diagnose a variety of diseases or conditions. For example,a malignant disease, a cardiovascular disease, an infectious disease, aninflammatory disease, an autoimmune disease, a metabolic disease, aneurological disease, or combinations of these diseases or conditions.

Where the disease or condition is a malignant disease, the bindingmolecule may specifically bind to a targeted tissue that includes anantigen selected from the group consisting of carcinoembryonic antigen,tenascin, epidermal growth factor receptor, platelet derived growthfactor receptor, fibroblast growth factor receptors, vascularendothelial growth factor receptors, gangliosides, HER/2neu receptorsand mixtures of these antigens. The targeted tissue may also include atumor. The binding molecule may specifically bind to antigens producedby or associated with the tumor including colon-specific antigen-p(CSAp), carcinoembryonic antigen (CEA), CD4, CD5, CD8, CD14, CD15, CD19,CD20, CD21, CD22, CD23, CD25, CD30, CD45, CD74, CD80, HLA-DR, Ia, Ii,MUC 1, MUC 2, MUC 3, MUC 4, NCA, EGFR, HER 2/neu, PAM-4, TAG-72, EGP-1,EGP-2, A3, KS-1, Le(y), S100, PSMA, PSA, tenascin, folate receptor,VEGF, P1GF, ILGF-1, necrosis antigens, IL-2, IL-6, T101, MAGE, andcombinations of these antigens. Particularly useful antigens includeCD74 and EGP-1, which may facilitate internalization of the boundantibody. Antibodies that recognize CD74 include LL1, the use of whichis described in U.S. Pat. No. 6,458,933; U.S. Pat. No. 6,395,276; U.S.Pat. No. 6,083,477; and U.S. 2003-0103982. Antibodies that recognizeEGP-1 include RS7, which is described in U.S. Ser. No. 10/377,121; U.S.Pat. No. 5,635,603; and Stein et al., 1990, Cancer Res., 50, 1330-1336.

The targeted tissue may include a multiple myleoma, a B-cell malignancy,or a T-cell malignancy. Specific B-cell malignancies may includeindolent forms of B-cell lymphomas, aggressive forms of B-celllymphomas, chronic leukemias, multiple myeloma, and acute lymphaticleukemias. The targeted tissue may also include a lymphoma such as anon-Hodgkin's lymphoma or a Hodgkin's lymphoma.

In addition, the targeted tissue(s) may include a solid tumor, such as amelanoma, a carcinoma, a sarcoma, a glioma, or combinations of thesemalignancies. Particular carcinomas may include esophageal, gastric,colonic, rectal, pancreatic, lung, breast, ovarian, urinary bladder,endometrial, cervical, testicular, renal, adrenal, liver cancer, orcombinations of these carcinomas.

The disease or condition may also include a cardiovascular disease thatis associated with granulocytes, lymphocytes, monocytes, D-dimer, and/orfibrin deposits. As such, the binding molecule (i.e., targetingmolecule) may specifically bind to antigens that are present ongranulocytes, lymphocytes, monocytes, and/or fibrin. Particularcardiovascular diseases or conditions may include a myocardialinfarction, ischemic heart disease, atherosclerotic plaques, fibrinclots, emboli, or a combinations of these disease or conditions.

The method may also be used to treat and/or diagnose infectiousdiseases, for example, bacterial disease, fungal disease, parasiticdisease, viral disease, protozoan disease, mycoplasmal, and combinationsof these infectious diseases. In particular, the infectious disease maybe caused by a pathogen selected from the group consisting ofMicrosporum, Trichophyton, Epidermophyton, Sporothrix schenckii,Cryptococcus neoformans, Coccidioides immitis, Histoplasma capsulatum,Blastomyces dermatitidis, Candida albicans, human immunodeficiency virus(HIV), herpes virus, cytomegalovirus, rabies virus, influenza virus,hepatitis B virus, Sendai virus, feline leukemia virus, Reovirus,poliovirus, human serum parvo-like virus, simian virus 40, respiratorysyncytial virus, mouse mammary tumor virus, Varicella-Zoster virus,Dengue virus, rubella virus, measles virus, adenovirus, human T-cellleukemia viruses, Epstein-Barr virus, murine leukemia virus, mumpsvirus, vesicular stomatitis virus, Sindbis virus, lymphocyticchoriomeningitis virus, wart virus, blue tongue virus, Anthrax bacillus,Streptococcus agalactiae, Legionella pneumophilia, Streptococcuspyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseriameningitidis, Pneumococcus, Hemophilis influenzae B, Treponema pallidum,Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae,Brucella abortus, Mycobacterium tuberculosis, Tetanus, a helminth, amalaria parasite, Plasmodium falciparum, Plasmodium vivax, Toxoplasmagondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosomarhodesiensei, Trypanosoma brucei, Schistosoma mansoni, Schistosomajapanicum, Babesia bovis, Elmeria tenella, Onchocerca volvulus,Leishmania tropica, Trichinella spiralis, Onchocerca volvulus, Theileriaparva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcusgranulosus, Mesocestoides corti, Mycoplasma arthritidis, Mycoplasmahyorhinis, Mycoplasma orale, Mycoplasma arginini, Acholeplasmalaidlawii, Mycoplasma salivarum, Mycoplasma pneumoniae, and combinationsof these pathogens.

The method may also be used to treat and/or diagnose autoimmune diseasesor conditions, such as acute idiopathic thrombocytopenic purpura,chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham'schorea, myasthenia gravis, systemic lupus erythematosus, lupusnephritis, rheumatic fever, polyglandular syndromes, bullous pemphigoid,diabetes mellitus, Henoch-Schonlein purpura,post-streptococcalnephritis, erythema nodosum, Takayasu's arteritis,Addison's disease, rheumatoid arthritis, multiple sclerosis,sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy,polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome,thromboangitisubiterans, Sjogren's syndrome, primary biliary cirrhosis,Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic activehepatitis, polymyositis/dermatomyositis, polychondritis, parnphigusvulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophiclateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia,perniciousanemia, rapidly progressive glomerulonephritis, psoriasis,fibrosing alveolitis, and combinations of these diseases or conditions.

Neurological diseases may also be treated or diagnosed by using themethod. For example, a neurological disease characterized by a metabolicdisorder, such as amyloidosis, may be treated or diagnosed by the methodwhere the targeted tissue includes an amyloid deposit.

In addition to administering the binding molecule, optionally theclearing agent, and the targetable molecule, the method may also includeadministering one or more additional therapeutic or diagnostic agents.Suitable therapeutic or diagnostic agents may include binding molecules(e.g., antibodies or fragments thereof), drugs, prodrugs, toxins,enzymes, enzyme-inhibitors, nucleases, hormones, hormone antagonists,immunomodulators, cytokines, chelators, boron compounds, uranium atoms,photoactive agents, radionuclides, and combinations of these agents. Theagents may be administering before, simultaneously, or afteradministration of the binding molecule, the optional clearing agent, andthe targetable molecule. Further, the agents may be conjugated to one ormore of the binding molecule, clearing agent, and/or the targetableconstruct. The agents may also be administered in combination with anemulsion or liposome, which may be conjugated to a compound such as thetargetable construct.

In one embodiment, the therapeutic agent includes a cytokine selectedfrom the group consisting of IL-1, IL-2, IL-3, IL-6, IL-10, IL-12,IL-18, IL-21, interferon-α, interferon-β, interferon-γ, G-CSF, andGM-CSF, and mixtures of these cytokines. In another embodiment, thetherapeutic agent includes an anti-angiogenic agent selected from thegroup consisting of angiostatin, endostatin, basculostatin, canstatin,maspin, anti-VEGF antibodies, anti-placental growth factor antibodies,anti-vascular growth factor antibodies, and mixtures of theseanti-angiogenic agents.

The method may include administering a diagnostic agent selected fromradioisotopes, dyes, radioopaque materials, contrast agents, fluorescentcompounds, enhancing agents, and combinations of these diagnosticagents.

It may be desirable to further administer a metal as a therapeutic ordiagnostic agent. For example, zinc, aluminum, gallium, lutetium,palladium, boron, gandolinium, uranium, manganese, iron, chrominum,copper, cobalt, nickel, dysprosium, rhenium, europium, terbium, holmium,neodymium, and combinations of these metals may be administered.

Paramagnetic ions, useful for diagnostic procedures, may also beadministered. Examples of paramagnetic ions include chromium (III),manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper(II), neodymium (III), samarium (III), ytterbium (III), gadolinium(III), vanadium (II), terbium (III), dysprosium (III), holmium (III),erbium (III), or combinations of these paramagnetic ions.

The therapeutic and/or diagnostic agent may include one or more agentsfor photodynamic therapy, (e.g., a photosensitizer). Photosensitizersmay include a benzoporphyrin monoacid ring A (BDP-MA), tin etiopurpurin(SnET2), sulfonated aluminum phthalocyanine (AlSPc) and lutetiumtexaphyrin (Lutex).

Therapeutic or diagnostic nuclides may also be administered, including¹⁸F, ³²P, ³³P, ⁴⁵Ti, ⁴⁷Sc, ⁵⁹Fe, ₆₂Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁵Se,⁷⁷As, ⁸⁶Y, ⁸⁹Zr, ⁹⁰Y, ⁹⁴Tc, ^(94m)Tc, ⁹⁹Mo, ^(99m)Tc, ¹⁰⁵Pd, ¹⁰⁵Rh,¹¹¹Ag, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵³Sm,¹⁵⁴⁻¹⁵⁸Gd, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁶⁹Er, ¹⁷⁵Lu, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re,¹⁸⁹Re, ¹⁹⁴Ir, ¹⁹⁸Au, ²¹¹At, ²¹¹Pb, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²²³Ra, ²²⁵Ac,and mixtures of these nuclides. Particularly suitable therapeuticnuclides may include ³²P, ³³P, ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁹⁰Y, ¹¹¹Ag,¹¹¹In, ¹²³I, ¹³¹I, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re,¹⁸⁸Re, ¹⁸⁹Re, ²¹¹At, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²²³Ra, ²²⁵Ac, or mixtures ofthese nuclides. Therapeutic nuclides may emit gamma particles and/orpositrons that have an energy of about 70 to about 700 keV.

Particularly suitable diagnostic nuclides may include ¹⁸F, ⁵²Fe, ⁶²Cu,⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr, ⁹⁴Tc, ^(94m)Tc, ^(99m)Tc, ¹¹¹In,¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, or mixtures of these nuclides. Diagnosticnuclides may emit gamma particles and/or positrons that have an energyof between about 25 to about 4000 keV.

The diagnostic agent may be useful when imaging methods are performed.For example, nuclides such as ¹⁸F may be included to perform positronemission tomography (PET). Alternatively, image enhancing agents usefulfor performing magnetic resonance imaging (MRI) may be included. Imageenhancing agents may include gadolinium ions, lanthanum ions, manganeseions, iron, chromium, copper, cobalt, nickel, fluorine, dysprosium,rhenium, europium, terbium, holmium, neodymium, or mixtures of theseagents. In another embodiment, one or more radiopaque agents or contrastagents for X-ray or computed tomography (CT) may be included. Radiopaqueor contrast agents may include barium, diatrizoate, ethiodized oil,gallium citrate, iocarmic acid, iocetamic acid, iodamide, iodipamide,iodoxamic acid, iogulamide, iohexol, iopamidol, iopanoic acid,ioprocemic acid, iosefamic acid, ioseric acid, iosulamide meglumine,iosemetic acid, iotasul, iotetric acid, iothalamic acid, iotroxic acid,ioxaglic acid, ioxotrizoic acid, ipodate, meglumine, metrizamide,metrizoate, propyliodone, thallous chloride, or combinations of theseagents.

The method may also include administering one or more ultrasoundcontrast agents such as a liposome or dextran. Liposomes may begas-filled.

The therapeutic and/or diagnostic method may also include performing anoperative, intravascular, laparoscopic, or endoscopic procedure, eitherbefore, simultaneously, or after the therapeutic and/or diagnosticmethod.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the structure of Bis In³⁺ IMP274.

FIG. 2 is a schematic representation of the structure of a SN-38analog/derivative of Bis In³⁺ IMP 274.

FIG. 3 is a schematic representation of the structure of a SN-38analog/derivative of Bis In³⁺ IMP 274 with SN-38 conjugated to acysteine by a penicillamine linkage.

FIG. 4 is a schematic representation of the structure of a SN-38analog/derivative of Bis In³⁺ IMP 274 with SN-38 conjugated to acysteine by a hindered ester linkage.

FIG. 5 is a schematic representation of the structure of IMP 225.

FIG. 6 is a schematic representation of the structure of Bis In³⁺ IMP224.

FIG. 7 is a graphic representation of the HPLC analysis (reverse phase)of In³⁺ IMP 274 after storage.

FIG. 8 is a graphic representation of the HPLC analysis (size exclusion)of In³⁺ IMP 274 after storage.

FIG. 9A and B are graphic representations of the HPLC analysis (reversephase) of In³⁺ IMP 274 incubated with mouse serum.

FIG. 10A and B are graphic representations of the HPLC analysis (reversephase) of In³⁺ IMP 274 incubated with human serum.

FIG. 11 is a graphic representation of the HPLC analysis (sizeexclusion) of In³⁺ IMP 274 incubated with mouse serum containing bsAb734 X hMN14.

FIG. 12 is a graphic representation of the HPLC analysis (sizeexclusion) of In³⁺ IMP 274 incubated with human serum containing bsAb734 X hMN14.

FIG. 13. is a graphic representation of the stability of IMP 294 (A) andIMP 295 (B) over a one week period. Samples were analyzed on day 0, 1,2, 3, 6, and 7.

FIG. 14 displays the results of a pre-targeting experiment using LL2×734bi-specific antibody and IMP-225 peptide in SCID mice inoculated withDaudi (Burkitt's lymphoma cells).

FIG. 15 is a schematic representation of a method for synthesizing aDTPA precursor and DTPA using a three step method.

FIG. 16 is a schematic representation of a method for synthesizing aDTPA precursor and DTPA using a four step method.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless otherwise specified, “a” or “an” means “one or more”.

“Predominantly” means “substantially” and/or at least 90%.

Overview

Disclosed herein are compounds that may be useful as targetableconstructs for therapeutic or diagnostic methods. The targetableconstruct may be specifically bound by a binding molecule such as abi-specific antibody (bsAb) or antibody fragment (bsFab), which has atleast one arm that binds the targetable construct and at least one otherarm that binds a targeted tissue. Desirably, the targetable constructincludes a peptide having at least two units of a recognizable hapten.Examples of recognizable haptens include, but are not limited to, DTPAand HSG. The targetable construct is conjugated to an effector molecule,which includes a variety of agents useful for treating or identifyingdiseased tissue. Examples of conjugated haptens and/or effectormolecules include, but are not limited to, chelators, metal chelatecomplexes, drugs, enzymes, and toxins (e.g., ricin, abrin, ribonuclease(e.g., RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviralprotein, gelonin, diphtherin toxin, Pseudomonas exotoxin, Pseudomonasendotoxin). Effector molecules may include lipids or polymers, which maybe associated with other effector molecules described herein. Forexample, lipids or polymers may form higher-ordered structures such asmicelles/liposomes or polymeric structures. Effector molecules mayinclude nanoparticles, which can be used to deliver effector moleculesas described herein.

Bi-specific antibody (bsAb) pretargeting represents a potentiallynon-immunogenic, highly selective alternative for diagnostic andtherapeutic applications. The bsAb pretargeting system described hereinrepresents an additional significant advantage over other pretargetingsystems in that it potentially can be developed for use with a varietyof different imaging or therapeutic agents. The flexibility of thissystem is based on use of an antibody directed against DTP or HSG andthe development of peptides containing the DTP or HSG residue.DTP-containing and/or HSG-containing peptides can be synthesized, andwhere the peptide contains DTP, the peptide can be labeled with chelatednuclides, such as ¹¹¹In, ⁹⁰Y, or ¹⁷⁷Lu, which may be useful in therapyor diagnosis. Antibodies have been generated against the DTPA-¹¹¹Inmoiety. For pretargeting, the selected peptides can be used incombination with bi-specific antibodies using the anti-DTPA-¹¹¹In Fab′fragment or the anti-HSG Fab′ fragment chemically stabilized with theFab′ fragment of either an anti-carcinoembryonic antigen antibody(anti-CEA) or an anti-colon-specific antigen-p antibody (anti-CSAp) toprovide tumor targeting capability for tumors expressing these antigens.However, other antigen targets may include diverse tumor-associatedantigens known in the art, such as against CD19, CD20, CD21, CD22, CD23,CD30, CD74, CD 80, HLA-DR, Ia, MUC 1, MUC 2, MUC 3, MUC 4, EGFR, HER2/neu, PAM-4, BrE3, TAG-72 (B72.3, CC49), EGP-1 (e.g., RS7), EGP-2(e.g., 17-1A and other Ep-CAM targets), Le(y) (e.g., B3), A3, KS-1,S100, IL-2, T101, necrosis antigens, folate receptors, angiogenesismarkers (e.g., VEGF), tenascin, PSMA, PSA, tumor-associated cytokines,MAGE and/or fragments thereof. Tissue-specific antibodies (e.g., againstbone marrow cells, such as CD34, CD74, etc., and parathyroglobulinantibodies, etc.) as well as antibodies against non-malignant diseasedtissues, such as fibrin and/or D-dimer of clots, macrophage antigens ofatherosclerotic plaques (e.g., CD74 antibodies), and also specificpathogen antibodies (e.g., against bacteria, viruses, and parasites) arewell known in the art.

The peptide described herein can be radiolabeled to a high specificactivity in a facile manner that avoids the need for purification. Invivo studies in tumor bearing nude mice showed the radiolabeled peptidescleared rapidly from the body with minimal retention in tumor or normaltissues. See, e.g., Tables 1-12, 14, and 16-18, which show that thepretargeting system is highly flexible, being capable of using a widearray of compounds of diagnostic imaging and therapeutic interest. Byachieving excellent tumor uptake and targeting ratios, the disclosedpretargeting system is highly promising for use in many applications.

Additionally encompassed is a method for detecting and/or treatingtarget cells, tissues or pathogens in a mammal, comprising administeringan effective amount of a binding molecule (e.g., a bi-specific antibodyor antibody fragment) comprising at least one arm that specificallybinds a targeted tissue and at least one other arm that specificallybinds a targetable construct. As used herein, the term “pathogen”includes, but is not limited to fungi (e.g., Microsporum, Trichophyton,Epidermophyton, Sporothrix schenckii, Cryptococcus neoformans,Coccidioides immitis, Histoplasma Capsulatum, Blastomyces dermatitidis,Candida albicans), viruses (e.g., human immunodeficiency virus (HW),herpes virus, cytomegalovirus, rabies virus, influenza virus, hepatitisB virus, Sendai virus, feline leukemia virus, Reo virus, polio virus,human serum parvo-like virus, simian virus 40, respiratory syncytialvirus, mouse mammary tumor virus, Varicella-Zoster virus, Dengue virus,rubella virus, measles virus, adenovirus, human T-cell leukemia viruses,Epstein-Barr virus, murine leukemia virus, mumps virus, vesicularstomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus,wart virus and blue tongue virus), parasites, bacteria (e.g., Anthraxbacillus, Streptococcus agalactiae, Legionella pneumophilia,Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae,Neisseria meningitidis, Pneumococcus, Hemophilis influenzae B, Treponemapallidum, Lyme disease spirochetes, Pseudomonas aeruginosa,Mycobacterium leprae, Brucella abortus, Mycobacterium tuberculosis andTetanus toxin), mycoplasma (e.g., Mycoplasma arthritidis, M. hyorhinis,M. orale, M. arginini, Acholeplasma laidlawii, M. salivarum, and M.pneumoniae) and protozoans (e.g., Plasmodium falciparum, Plasmodiumvivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi,Trypanosoma rhodesiensei, Trypanosoma brucei, Schistosoma mansoni,Schistosoma japanicum, Babesia bovis, Eimeria tenella, Onchocercavolvulus, Leishmania tropica, Trichinella spiralis, Onchocerca volvulus,Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata,Echinococcus granulosus and Mesocestoides corti). See U.S. Pat. No.5,332,567.

Also disclosed herein are binding molecules which include antibodies andantibody fragments. The antibody fragments are antigen binding portionsof an antibody, such as Fab or F(ab)₂ and the like. The antibodyfragments bind to the same antigen that is recognized by the intactantibody. For example, an anti-CD22 monoclonal antibody fragment bindsto an epitope of CD22.

The term “antibody fragment” also includes any synthetic or geneticallyengineered protein that acts like an antibody by binding to a specificantigen to form a complex. For example, antibody fragments includeisolated fragments, “Fv” fragments, consisting of the variable regionsof the heavy and light chains, recombinant single chain polypeptidemolecules in which light and heavy chain variable regions are connectedby a peptide linker (“sFv proteins”), and minimal recognition unitsconsisting of the amino acid residues that mimic the “hypervariableregion.” Three of these so-called “hypervariable” regions or“complementarity-determining regions” (CDR) are found in each variableregion of the light or heavy chain. Each CDR is flanked by relativelyconserved framework regions (FR). The FR are thought to maintain thestructural integrity of the variable region. The CDRs of a light chainand the CDRs of a corresponding heavy chain form the antigen-bindingsite. The “hypervariability” of the CDRs accounts for the diversity ofspecificity of antibodies.

As used herein, the term “subject” and “patient” refer to any animal(i.e., vertebrates and invertebrates) including, but not limited tohumans and other primates, rodents (e.g., mice, rats, and guinea pigs),lagamorphs (e.g., rabbits), bovines (e.g., cattle), ovines (e.g.,sheep), caprines (e.g., goats), porcines (e.g., swine), equines (e.g.,horses), canines (e.g., dogs), felines (e.g., cats), domestic fowl(e.g., chickens, turkeys, ducks, geese, other gallinaceous birds, etc.),as well as feral or wild animals, including, but not limited to, suchanimals as ungulates (e.g., deer), bear, fish, lagamorphs, rodents,birds, etc. It is not intended that the term be limited to a particularage or sex. Thus, adult and newborn subjects, as well as fetuses,whether male or female, are encompassed by the term.

Constructs Targetable to Antibodies

As noted, the above-described compounds can be used as targetableconstructs. The targetable construct can be of diverse structure, but isselected not only to diminish the elicitation of immune responses, butalso for rapid in vivo clearance when used within the bsAb targetingmethod. Hydrophobic agents are best at eliciting strong immuneresponses, whereas hydrophilic agents are preferred for rapid in vivoclearance, thus, an ideal construct will possess both hydrophobic andhydrophilic qualities. This is accomplished, in part, by relying on theuse of hydrophilic chelating agents (such as DTPA) to offset theinherent hydrophobicity of many organic effectors (e.g., toxins such ascamptothecin). Also, sub-units of the targetable construct may be chosenwhich have opposite solution properties, for example, peptides, whichcontain amino acids, some of which are hydrophobic and some of which arehydrophilic. Aside from peptides, carbohydrates may also be used orother suitable molecules may be used to synthesize the compoundsdescribed herein.

The targetable construct may include a peptide backbone (e.g., as aspacer) having as few as two amino-acid residues, (with preferably twoto ten amino acid residues), and the backbone may be coupled to othermoieties such as chelating agents. The targetable construct should be alow molecular weight construct, preferably having a molecular weight ofless than 50,000 daltons, and advantageously less than about 20,000daltons, 10,000 daltons or 5,000 daltons, including any metal ions thatmay be bound to the chelating agents. For instance, the known peptideDTPA-Tyr-Lys(DTPA)-OH (wherein DTPA is diethylenetriaminepentaaceticacid) has been used to generate antibodies against the indium-DTPAportion of the molecule, as noted above. However, by use of thenon-indium-containing molecule, and appropriate screening steps, new Absagainst the tyrosyl-lysine dipeptide can also be made. More usually, theantigenic peptide of the targetable construct will have four or moreresidues, such as the peptide N-acetyl-Cys-Lys(DTPA)-Tyr-Lys(DTPA)-NH₂(SEQ ID NO:1).

The haptens of the targetable construct also provide an immunogenicrecognition moiety. Using a hapten such as a DTPA or HSG hapten, bsAbswith high specificity for the construct can be generated. This occursbecause antibodies raised to the DTPA or HSG hapten are known and can beeasily incorporated into the appropriate bsAb. Thus, coupling of thehaptens to the peptide backbone would result in a targetable constructthat is specifically recognized by the bsAb or bsFab.

The compound may incorporate unnatural amino acids, e.g., D-amino acids,into a peptide backbone structure to ensure that, when used with thefinal bsAb/construct system, the arm of the bsAb which recognizes thetargetable construct is completely specific. Further, other backbonestructures such as those constructed from other non-natural amino acidsand peptoids may be present in the compound. Incorporation of D-aminoacids and/or L-amino acids can also be used to control the stability ofa peptide

Peptides to be used as immunogens are synthesized conveniently on anautomated peptide synthesizer using a solid-phase support and standardtechniques of repetitive orthogonal deprotection and coupling. Freeamino groups in the peptide, that are to be used later for chelateconjugation, are advantageously blocked with standard protecting groupssuch as an acetyl group. Such protecting groups will be known to theskilled artisan. See Greene and Wuts Protective Groups in OrganicSynthesis, 1999 (John Wiley and Sons, N.Y.). When the peptides areprepared for later use within the bsAb system, they are advantageouslycleaved from the resins to generate the corresponding C-terminal amides,in order to inhibit in vivo carboxypeptidase activity. Methods forpreparing targetable constructs are described in U.S. patent applicationSer. Nos. 09/337,756; 09/382,186; 09/823,746; and 10/150,654; all ofwhich are incorporated herein by reference.

Chelate Moieties

The presence of hydrophilic chelate moieties on the targetable constructhelps to ensure rapid in vivo clearance. In addition to hydrophilicity,chelators are chosen for their metal-binding properties, and may bechanged at will because, at least for those targetable constructs forwhich the bsAb epitope is not the chelator, recognition of themetal-chelate complex is not required.

Particularly useful metal-chelate combinations include 2-benzyl-DTPA andits monomethyl and cyclohexyl analogs, used with ⁴⁷Sc, ⁵²Fe, ⁶⁷Ga, ⁶⁸Ga,¹¹¹In, ⁸⁹Zr, ⁹⁰Y, ¹⁶¹Tb, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, and ²²⁵Ac forradio-imaging and RAIT. The same chelators, when complexed withnon-radioactive metals such as Mn, Fe and Gd for use with MRI, may beused along with the bsAbs of the methods described herein. Macrocyclicchelators such as NOTA (1,4,7-triaza-cyclononane-N,N,N-triacetic acid),DOTA, and TETA (p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid)are of use with a variety of metals and radiometals, most particularlywith radionuclides of Ga, Y and Cu, respectively.

DTPA and DOTA-type chelators, where the ligand includes hard basechelating functions, such as carboxylate or amine groups, are mosteffective for chelating hard acid cations, especially Group IIa andGroup IIIa metal cations. Such metal-chelate complexes can be made verystable by tailoring the ring size to the metal of interest. Otherring-type chelators such as macrocyclic polyethers are of interest forstably binding nuclides such as ²²³Ra for RAIT. Porphyrin chelators maybe used with numerous radiometals, and are also useful as certain coldmetal complexes for bsAb-directed immuno-phototherapy. Also, more thanone type of chelator may be conjugated to the targetable construct tobind multiple metal ions, e.g., cold ions, diagnostic radionuclidesand/or therapeutic radionuclides.

Particularly useful diagnostic radionuclides that can be bound to thechelating agents of the targetable construct include, but are notlimited to, ¹¹⁰In, ¹¹¹In, ¹⁷⁷Lu, ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga,⁶⁸Ga, ⁸⁶Y, ⁹⁰Y, ⁸⁹Zr, ^(94m)Tc, ⁹⁴Tc, ^(99m)Tc, ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I,¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd, ³²P, ¹¹C, ¹³N, ¹⁵O, ¹⁸⁶Re, ¹⁸⁸Re, ⁵¹Mn, ⁵⁵Co, ⁷²As,⁷⁵Br, ^(82m)Rb, ⁸³Sr, or other gamma-, beta-, or positron-emitters.Preferably, the diagnostic radionuclides include a decay energy in therange of 25 to 10,000 keV, more preferably in the range of 25 to 4,000keV, and even more preferably in the range of 20 to 1,000 keV, and stillmore preferably in the range of 70 to 700 keV. Total decay energies ofuseful positron-emitting radionuclides are preferably <2,000 keV, morepreferably under 1,000 keV, and most preferably <700 keV. Radionuclidesuseful as diagnostic agents utilizing gamma-ray detection include, butare not limited to: ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁶⁷Cu, ⁶⁷Ga, ⁷⁵Se, ⁹⁷Ru,^(99m)Tc, ¹¹¹In, ^(114m)In, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁶⁹Yb, ¹⁹⁷Hg, and ²⁰¹Tl.Decay energies of useful gamma-ray emitting radionuclides are preferably20-2000 keV, more preferably 60-600 keV, and most preferably 100-300keV.

Particularly useful therapeutic radionuclides that can be bound to thechelating agents of the targetable construct include, but are notlimited to, ¹¹¹In, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁹⁰Y,¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy,¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr,⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, and ²¹¹Pb.The therapeutic radionuclide preferably has a decay energy in the rangeof 25 to 10,000 keV. Decay energies of useful beta-particle-emittingnuclides are preferably 25-5,000 keV, more preferably 100-4,000 keV, andmost preferably 500-2,500 keV. Also preferred are radionuclides thatsubstantially decay with Auger-emitting particles. For example, ⁵⁸Co,⁶⁷Ga, ^(88m)Br, ^(99m)Tc, ^(103m)Rh, ¹⁰⁹Pt, ¹¹¹In, ¹¹⁹Sb, ¹²⁵I, ¹⁶¹Ho,189m Os and ¹⁹²Ir. Decay energies of useful beta-particle-emittingnuclides are preferably <1,000 keV, more preferably <100 keV, and mostpreferably <70 keV. Also preferred are radionuclides that substantiallydecay with generation of alpha-particles. Such radionuclides include,but are not limited to: ¹⁵²Dy, ²¹¹At, ²¹²Bi, ²²³Ra, ²¹⁹Rn, ²¹⁵Po, ²¹¹Bi,²²⁵Ac, ²²¹Fr, ²¹⁷At, ²¹³Bi and ²²⁵Fm. Decay energies of usefulalpha-particle-emitting radionuclides are preferably 2,000-9,000 keV,more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.

Chelators such as those disclosed in U.S. Pat. No. 5,753,206, especiallythiosemi-carbazonylglyoxylcysteine (Tscg-Cys) andthiosemicarbazinyl-acetylcysteine (Tsca-Cys) chelators areadvantageously used to bind soft acid cations of Tc, Re, Bi and othertransition metals, lanthanides and actinides that are tightly bound tosoft base ligands, especially sulfur- or phosphorus-containing ligands.It may be useful to link more than one type of chelator to a peptide,(e.g., a hard acid chelator like DTPA for In(III) cations, and a softacid chelator like Tscg-Cys for Tc cations). Because antibodies to adi-DTPA hapten are known (Barbet '395, supra) and are readily coupled toa targeting antibody to form a bsAb, it is possible to use a peptidewith a cold di-DTPA chelator (e.g., not chelated with a radioisotope)and a chelator with a radioisotope in a pretargeting protocol fortargeting the radioisotope to diseased tissue. One example of such apeptide is Ac-Lys(DTPA)-Tyr-Lys(DTPA)-Lys(Tscg-Cys)-NH₂ (SEQ ID NO:2).This peptide can be preloaded with In(III) and then labeled with^(99m)Tc cations, the In(III) ions being preferentially chelated by theDTPA and the Tc cations binding preferentially to the thiol-containingTscg-Cys. Other hard acid chelators such as NOTA, DOTA, TETA and thelike can be substituted for the DTPA groups, and Mabs specific to themcan be produced using analogous techniques to those used to generate theanti-di-DTPA Mab.

It will be appreciated that two different hard acid or soft acidchelators can be incorporated into the linker, (e.g., with differentchelate ring sizes), to bind preferentially to two different hard acidor soft acid cations, based on the differing sizes of the cations, thegeometries of the chelate rings, and the preferred complex ionstructures of the cations. This will permit two different metals, one orboth of which may be radioactive or useful for MRI enhancement, to beincorporated into a linker for eventual capture by a pretargeted bsAb.

Chelators are coupled to the peptides of the targetable construct usingstandard chemistries, some of which are discussed more fully in theworking examples below. See also Karacay et al. Bioconjugate Chem.11:842-854 (2000); and U.S. patent application Ser. Nos. 09/337,756;09/382,186; 09/823,746; and 10/150,654; all of which are incorporatedherein by reference. The protecting group abbreviations “Aloc” and“Fmoc” used herein refer to the groups allyloxycarbonyl andfluorenylmethyloxy carbonyl.

General Methods for Preparation of Metal Chelates

Chelator-peptide conjugates may be stored for long periods as solids.They may be metered into unit doses for metal-binding reactions, andstored as unit doses either as solids, aqueous or semi-aqueoussolutions, frozen solutions or lyophilized preparations. They may belabeled by well-known procedures.

Typically, a hard acid cation is introduced as a solution of aconvenient salt, and is taken up by the hard acid chelator and possiblyby the soft acid chelator. However, later addition of soft acid cationsleads to binding thereof by the soft acid chelator, displacing any hardacid cations which may be chelated therein. For example, even in thepresence of an excess of cold ¹¹¹InCl₃, a soft acid chelator may belabeled quantitatively with Tc cations provided by ^(99m)Tc(V)glucoheptonate or generated in situ with stannous chloride and^(99m)Na—TcO₄.

Other soft acid cations such as ¹⁸⁶Re, ¹⁸⁸Re, ²¹³Bi and divalent ortrivalent cations of Mn, Co, Ni, Pb, Cu, Cd, Au, Fe, Ag (monovalent), Znand Hg, especially ⁶⁴Cu and ⁶⁷Cu, and the like, some of which are usefulfor radioimmunodetection or radioimmunotherapy, may be loaded onto thelinker peptide by analogous methods. Rhenium cations also can begenerated in situ from perrhenate and stannous ions or a prereducedrhenium glucoheptonate or other transchelator can be used. Becausereduction of perrhenate requires more stannous ion (typically above 200g/mL final concentration) than is needed for the reduction of Tc, extracare needs to be taken to ensure that the higher levels of stannous iondo not reduce sensitive disulfide bonds such as those present indisulfide-cyclized peptides. During radiolabeling with rhenium, similarprocedures are used as are used with the ^(99m)Tc. One method for thepreparation of ReO metal complexes of the Tscg-Cys ligands is byreacting the peptide with ReOCl₃(P(Ph₃)₂ but it is also possible to useother reduced species such as ReO(ethylenediamine)₂.

Other methods for preparing metal-chelate complexes are described inU.S. patent application Ser. Nos. 09/337,756; 09/382,186; 09/823,746;and 10/150,654; all of which are incorporated herein by reference.

Methods of Administering Targetable Constructs, bsAbs, and AdditionalTherapeutic or Diagnostic Agents

It should be noted that much of the discussion presented hereinbelowfocuses on the use of bi-specific antibodies and targetable constructsin the context of treating diseased tissue. However, also contemplatedis the use of the targetable constructs and bi-specific antibodies intreating and/or imaging normal tissue and organs using the methodsdescribed in U.S. Pat. Nos. 6,126,916; 6,077,499; 6,010,680; 5,776,095;5,776,094; 5,776,093; 5,772,981; 5,753,206; 5,746,996; 5,697,902;5,328,679; 5,128,119; 5,101,827; and 4,735,210, which are incorporatedherein by reference. As used herein, the term “tissue” refers totissues, including but not limited to, tissues from the ovary, thymus,parathyroid, bone marrow or spleen. An important use when targetingnormal tissues is to identify and treat them when they are ectopic(i.e., displaced from their normal location), such as in endometriosis.

The targetable construct and/or bsAb may be administered intravenously,intraarterially, intraoperatively, endoscopically, intraperitoneally,intramuscularly, subcutaneously, intrapleurally, intrathecally, byperfusion through a regional catheter, or by direct intralesionalinjection, orally, and can be by continuous infusion or by single ormultiple boluses or through other methods known to those skilled in theart for diagnosing (detecting) and treating diseased tissue. Further,the targetable construct may include agents for other methods ofdetecting and treating diseased tissue including, without limitation,conjugating dextran or liposome formulations to the targetable constructfor use with ultrasound, or other contrast agents for use with otherimaging modalities, such as X-ray, CT, PET, SPECT and ultrasound, aspreviously described.

The administration of a bsAb and the targetable construct discussedabove may be conducted by administering the bsAb at some time prior toadministration of the therapeutic agent (i.e., effector) which isassociated with the linker moiety. The doses and timing of the reagentscan be readily devised by a skilled artisan, and are dependent on thespecific nature of the reagents employed. If a bsAb-F(ab′)₂ derivativeis given first, then a waiting time of 1-6 days before administration ofthe targetable construct may be appropriate. If an IgG-Fab′ bsAbconjugate is the primary targeting vector, then a longer waiting periodbefore administration of the linker moiety may be indicated, in therange of 3-15 days. Alternatively, the bsAb and the targetable constructmay be administered substantially at the same time in either a cocktailform or by administering one after the other.

A wide variety of diagnostic and therapeutic reagents can beadvantageously conjugated to the targetable construct. Generally,diagnostic and therapeutic agents can include isotopes, drugs, toxins,oligonucleotides (e.g., antisense oligonucleotides and interferenceRNAs), cytokines, conjugates with cytokines, hormones, growth factors,conjugates, radionuclides, contrast agents, metals, cytotoxic drugs, andimmune modulators. For example, gadolinium metal is used for magneticresonance imaging and fluorochromes can be conjugated for photodynamictherapy. Moreover, contrast agents can be MRI contrast agents, such asgadolinium ions, lanthanum ions, manganese ions, iron, chromium, copper,cobalt, nickel, dysprosium, rhenium, europium, terbium, holmium,neodymium or other comparable label, CT contrast agents, and ultrasoundcontrast agents. Additional diagnostic agents can include fluorescentlabeling compounds such as fluorescein isothiocyanate, rhodamine,phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde andfluorescamine, chemiluminescent compounds including luminol, isoluminol,an aromatic acridinium ester, an imidazole, an acridinium salt and anoxalate ester, and bioluminescent compounds including luciferin,luciferase and aequorin. Radionuclides can also be used as diagnosticand/or therapeutic agents, including for example, ⁹⁰Y, ¹¹¹In, ¹³¹I,^(99m)Tc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁷⁷Lu, ⁶⁷Cu, ²¹²Bi, ²¹³Bi, and ²¹¹At.

Therapeutic agents also include, for example, chemotherapeutic drugssuch as vinca alkaloids, anthracyclines, epidophyllotoxins, taxanes,antimetabolites, alkylating agents, antibiotics, Cox-2 inhibitors,antimitotics, antiangiogenic and apoptotoic agents, particularlydoxorubicin, methotrexate, taxol, CPT-11, camptothecins, and others fromthese and other classes of anticancer agents. Conjugation ofcamptothecins to Poly-(L-Glutamic Acid) has been described. See Singeret al., Annals of N.Y. Acad. of Sci., 2000;922:136-500. Other usefultherapeutic agents for the preparation of immunoconjugates and antibodyfusion proteins include nitrogen mustards, alkyl sulfonates,nitrosoureas, triazenes, folic acid analogs, COX-2 inhibitors,pyrimidine analogs, purine analogs, platinum coordination complexes,hormones, and the like. Suitable therapeutic agents are described inREMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co.1995), and in GOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OFTHERAPEUTICS, 7th Ed. (MacMillan Publishing Co. 1985), as well asrevised editions of these publications. Other suitable therapeuticagents, such as experimental drugs, are known to those of skill in theart. Therapeutic agents may also include, without limitation, othersdrugs, prodrugs and/or toxins. The terms “drug,” “prodrug,” and “toxin”are defined throughout the specification. The terms “diagnostic agent”or “diagnosis” include, but are not limited to, detection agent,detection, or localization. The therapeutic and diagnostic agents may beassociated with lipids capable of forming emulsions or liposomes orpolymers capable of forming polymeric structures.

When the targetable construct includes a diagnostic agent, the bsAb ispreferably administered prior to administration of the targetableconstruct (which includes the diagnostic agent). After sufficient timehas passed for the bsAb to target to the diseased tissue, the targetableconstruct including the diagnostic agent (i.e., effector) isadministered, so that imaging can be performed. Tumors can be detectedin body cavities by means of directly or indirectly viewing variousstructures to which light of the appropriate wavelength is delivered andthen collected, or even by special detectors, such as radiation probesor fluorescent detectors, and the like. Lesions at any body site can beviewed so long as nonionizing radiation can be delivered and recapturedfrom these structures. For example, PET which is a high resolution,non-invasive, imaging technique can be used with antibodies andtargetable constructs for the visualization of human disease. In PET,511 keV gamma photons produced during positron annihilation decay aredetected. X-ray, computed tomography (CT), MRI and gamma imaging (e.g.,Single Photon Emission Computed Tomography (SPECT)) may also be utilizedthrough use of a diagnostic agent that functions with these modalities.As discussed earlier, the targetable construct may include radioactivediagnostic agents that emit 25-10,000 keV gamma-, beta-, alpha- andauger- particles and/or positrons. Examples of such agents include, butare not limited to ¹⁸F, ⁴⁵Ti, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr,^(94m)Tc, ⁹⁴Tc, ^(99m)Tc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd and¹⁷⁵Lu.

The present bsAbs or bsFabs can be used in a method of photodynamictherapy (PDT) as discussed in U.S. Pat. Nos. 6,096,289; 4,331,647;4,818,709; 4,348,376; 4,361,544; 4,444,744; 5,851,527. In PDT, aphotosensitizer, e.g., a hematoporphyrin derivative such asdihematoporphyrin ether, is administered to a subject. Anti-tumoractivity is initiated by the use of light, e.g., 630 nm. Alternatephotosensitizers can be utilized, including those useful at longerwavelengths, where skin is less photosensitized by the sun. Examples ofsuch photosensitizers include, but are not limited to, benzoporphyrinmonoacid ring A (BPD-MA), tin etiopurpurin (SnET2), sulfonated aluminumphthalocyanine (AlSPc) and lutetium texaphyrin (Lutex).

Additionally, in PDT, a diagnostic agent may be injected, for example,systemically, and laser-induced fluorescence can be used by endoscopesincluding wireless capsule-sized endoscopes or cameras to detect sitesof cancer which have accreted the light-activated agent. For example,this has been applied to fluorescence bronchoscopic disclosure of earlylung tumors. Doiron et al. Chest 76:32 (1979). In another example, theantibodies and antibody fragments can be used in single photon emission.For example, a Tc-99m-labeled diagnostic agent can be administered to asubject following administration of antibodies or antibody fragments.The subject is then scanned with a gamma camera which producessingle-photon emission computed tomographic images and defines thelesion or tumor site.

Photoactive agents or dyes may be useful as therapeutic and/ordiagnostic reagents. For example, therapeutically usefulimmunoconjugates can be obtained by conjugating photoactive agents ordyes to an antibody composite. Fluorescent and other chromogens, ordyes, such as porphyrins sensitive to visible light, have been used todetect and to treat lesions by directing the suitable light to thelesion. In therapy, this has been termed photoradiation, phototherapy,or photodynamic therapy (Jori et aL (eds.), Photodynamic Therapy ofTumors and Other Diseases (Libreria Progetto 1985); van den Bergh, Chem.Britain 22:430 (1986)). Moreover, monoclonal antibodies have beencoupled with photoactivated dyes for achieving phototherapy. Mew et al.,J. Immunol. 130:1473 (1983); idem., Cancer Res. 45:4380 (1985); Oseroffet al., Proc. Natl. Acad. Sci. USA 83:8744 (1986); idem., Photochem.Photobiol. 46:83 (1987); Hasan et al., Prog. Clin. Biol. Res. 288:471(1989); Tatsuta et al., Lasers Surg. Med. 9:422 (1989); Pelegrin et al.,Cancer 67:2529 (1991). However, these earlier studies did not includeuse of endoscopic therapy applications, especially with the use ofantibody fragments or subfragments. Thus, the immunoconjugates mayinclude photoactive agents or dyes. Endoscopic methods of detection andtherapy are described in U.S. Pat. Nos. 4,932,412; 5,525,338; 5,716,595;5,736,119; 5,922,302; 6,096,289; and 6,387,350, which are incorporatedherein by reference in their entirety.

Radiopaque and contrast materials are used for enhancing X-rays andcomputed tomography, and include iodine compounds, barium compounds,gallium compounds, thallium compounds, etc. Specific compounds includebarium, diatrizoate, ethiodized oil, gallium citrate, iocarmic acid,iocetamic acid, iodamide, iodipamide, iodoxamic acid, iogulamide,iohexol, iopamidol, iopanoic acid, ioprocemic acid, iosefamic acid,ioseric acid, iosulamide meglumine, iosemetic acid, iotasul, iotetricacid, iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid,ipodate, meglumine, metrizamide, metrizoate, propyliodone, and thallouschloride. Ultrasound contrast material may also by used includingdextran and liposomes, particularly gas-filled liposomes.

Administering Immunomodulators

In one embodiment, an immunomodulator, such as a cytokine, may also beconjugated to the targetable construct by a linker or through othermethods known by those skilled in the art. As used herein, the term“immunomodulator” includes cytokines, stem cell growth factors,lymphotoxins, such as tumor necrosis factor (TNF), and hematopoieticfactors, such as interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3,IL-6, IL-10, IL-12, IL-18, and IL-21), colony stimulating factors (e.g.,granulocyte-colony stimulating factor (G-CSF) and granulocytemacrophage-colony stimulating factor (GM-CSF)), interferons (e.g.,interferons-α, -β and -γ), the stem cell growth factor designated “S1factor,” erythropoietin and thrombopoietin. Examples of suitableimmunomodulator moieties include IL-2, IL-6, IL-10, IL-12, IL-18, IL-21,interferons, TNFs (e.g., TNF-α), and the like.

Administering Drugs and Prodrugs

Certain cytotoxic drugs that are useful for anticancer therapy arerelatively insoluble in serum. In addition, some cytotoxic drugs arealso quite toxic in an unconjugated form, and their toxicity isconsiderably reduced by conversion to prodrugs. Conversion of a poorlysoluble drug to a more soluble conjugate, e.g., a glucuronide, an esterof a hydrophilic acid or an amide of a hydrophilic amine, will improveits solubility in the aqueous phase of serum and its ability to passthrough venous, arterial or capillary cell walls and to reach theinterstitial fluid bathing the tumor. Cleavage of the prodrug depositsthe less soluble drug at the target site. Many examples of suchprodrug-to-drug conversions are disclosed in U.S. Pat. No. 5,851,527, toHansen.

Conversion of certain toxic substances such as aromatic or alicyclicalcohols, thiols, phenols and amines to glucuronides in the liver is thebody's method of detoxifying them and making them more easily excretedin the urine. One type of antitumor drug that can be converted to such asubstrate is epirubicin, a 4-epimer of doxorubicin (Adriamycin), whichis an anthracycline glycoside and has been shown to be a substrate forhuman beta-D-glucuronidase. See, e.g., Arcamone Cancer Res. 45:5995(1985). Other analogues with fewer polar groups are expected to be morelipophilic and show greater promise for such an approach. Other drugs ortoxins with aromatic or alicyclic alcohol, thiol or amine groups arecandidates for such conjugate formation. These drugs, or other prodrugforms thereof, are suitable candidates for the site-specific enhancementmethods of the presently described compounds and methods.

The prodrug CPT-11 (irinotecan) is converted in vivo by carboxylesteraseto the active metabolite SN-38. One application of the therapeuticmethod, therefore, is to use a bsAb targeted against a tumor and ahapten (e.g. di-DTPA) followed by injection of adi-DTPA-carboxylesterase conjugate. Once a suitable tumor-to-backgroundlocalization ratio has been achieved, the CPT-11 is given and thetumor-localized carboxylesterase serves to convert CPT-11 to SN-38 atthe tumor. Due to its poor solubility, the active SN-38 will remain inthe vicinity of the tumor and, consequently, will exert an effect onadjacent tumor cells that are negative for the antigen being targeted.This is a further advantage of the method. Modified forms ofcarboxylesterases have been described and are within the scope of thedisclosed compounds and methods. See, e.g., Potter et al., Cancer Res.58:2646-2651 (1998) and Potter et al., Cancer Res. 58:3627-3632 (1998).In another embodiment, CPT-11 may be conjugated to a targetableconstruct that includes DTPA or a targeting molecule, which can furtherenhance localization and activation of CPT-11 to SN-38 at the tumor.

Etoposide is a widely used cancer drug that is detoxified to a majorextent by formation of its glucuronide and is within the scope of thedisclosed compounds and methods. See, e.g., Hande et al. Cancer Res.48:1829-1834 (1988). Glucuronide conjugates can be prepared fromcytotoxic drugs and can be injected as therapeutics for tumorspre-targeted with mAb-glucuronidase conjugates. See, e.g., Wang et al.Cancer Res. 52:4484-4491 (1992). Accordingly, such conjugates also canbe used with the pre-targeting approach described here. Similarly,designed prodrugs based on derivatives of daunomycin and doxorubicinhave been described for use with carboxylesterases and glucuronidases.See, e.g., Bakina et al. J. Med Chem. 40:4013-4018 (1997). Otherexamples of prodrug/enzyme pairs that can be used within the presentmethods include, but are not limited to, glucuronide prodrugs of hydroxyderivatives of phenol mustards and beta-glucuronidase; phenol mustardsor CPT-11 and carboxypeptidase; methotrexate-substituted alpha-aminoacids and carboxypeptidase A; penicillin or cephalosporin conjugates ofdrugs such as 6-mercaptopurine and doxorubicin and beta-lactamase;etoposide phosphate and alkaline phosphatase.

Co-administering Enzymes and Prodrugs

An enzyme capable of activating a prodrug at the target site orimproving the efficacy of a normal therapeutic by controlling the body'sdetoxification pathways may be a component of the compound (e.g.,conjugated to the spacer or hapten). An enzyme-hapten conjugate can beadministered to the subject following administration of thepre-targeting bsAb and can be directed to the target site. After theenzyme is localized at the target site, a cytotoxic drug is injected,which is known to act at the target site, or a prodrug form thereofwhich is converted to the drug in situ by the pretargeted enzyme. Afterbeing administered, the drug may be detoxified to form an intermediateof lower toxicity, most commonly a glucuronide, using the mammal'sordinary detoxification processes. The detoxified intermediate, e.g.,the glucuronide, is reconverted to its more toxic form by thepretargeted enzyme and thus has enhanced cytotoxicity at the targetsite. This results in a recycling of the drug. Similarly, anadministered prodrug can be converted to an active drug through normalbiological processes. The pretargeted enzyme improves the efficacy ofthe treatment by recycling the detoxified drug. This approach can beadopted for use with any enzyme-drug pair.

In an alternative embodiment, the enzyme-hapten conjugate can be mixedwith the targeting bsAb prior to administration to the patient. After asufficient time has passed for the enzyme-hapten-bsAb conjugate tolocalize to the target site and for unbound conjugate to clear fromcirculation, a prodrug is administered. As discussed above, the prodrugis then converted to the drug in situ by the pre-targeted enzyme.

In another embodiment, the pre-targeting bsAb is administered to thepatient and allowed to localize to the target and substantially clearcirculation. At an appropriate later time, a targetable constructcomprising a prodrug, for example poly-glutamic acid (SN-38-ester)₁₀, isgiven, thereby localizing the prodrug specifically at the tumor target.It is known that tumors have increased amounts of enzymes released fromintracellular sources due to the high rate of lysis of cells within andaround tumors. A practitioner can exploit this characteristic byappropriately selecting prodrugs capable of being activated by theseenzymes. For example, carboxylesterase activates the prodrugpoly-glutamic acid (SN-38-ester)₁₀ by cleaving the ester bond of thepoly-glutamic acid (SN-38-ester)₁₀ releasing large concentrations offree SN-38 at the tumor. Alternatively, the appropriate enzyme also canbe targeted to the tumor site.

After cleavage from the targetable construct, the drug is internalizedby the tumor cells. Alternatively, the drug can be internalized as partof an intact complex by virtue of cross-linking at the target. Thetargetable construct can induce internalization of tumor-bound bsAb andthereby improve the efficacy of the treatment by causing higher levelsof the drug to be internalized.

Compounds that Include Prodrugs Conjugated to Peptide Carriers

A variety of peptide carriers (e.g., as spacers) are well-suited forconjugation to prodrugs, including polyamino acids, such as polylysine,polyglutamic (E) and aspartic acids (D), including D-amino acid analogsof the same, and co-polymers, such as poly(Lys-Glu) {poly[KE]},advantageously at a ratio from 1:10 to 10:1. Copolymers based on aminoacid mixtures such as poly(Lys-Ala-Glu-Tyr (SEQ ID NO: 3) (KAEY;5:6:2:1) can also be employed. Smaller polymeric carriers of definedmolecular weight can be synthesized by solid-phase peptide synthesistechniques, readily producing polypeptides of from 2-50 residues inchain length. Another advantage of this type of reagent, other thanprecise structural definition, is the ability to place single or anydesired number of chemical handles at certain points in the chain. Thesecan be used later for attachment of recognition and therapeutic haptensat chosen levels of each moiety.

Poly(ethylene) glycol [PEG] has desirable in vivo properties for abi-specific antibody prodrug approach. The desirable in vivo propertiesof PEG derivatives and the limited loading capacity due to their dimericfunctionality has led to the preparation of PEG co-polymers havinggreater hapten-bearing capacity such as those described by Poiani et al.See, e.g., Poiani et al. Bioconjugate Chem., 5:621-630, 1994. PEG can beused to conjugate any component of the compound, (such as drugs orprodrugs to lysine residues). For example, PEG derivatives can beactivated at both ends to create bis(succinimidyl)carbonate derivativesand co-polymerized with multi-functional diamines such as lysine. Theproduct of such co-polymerization, containing(-Lys(COOH)-PEG-Lys(COOH)-PEG-)_(n) repeat units wherein the lysylcarboxyl group is not involved in the polymerization process, can beused for attachment of SN-38 residues. The SN-38 residues are reactedwith the free carboxyl groups to produce SN-38 esters of the(-Lys-(COOH)-PEG-Lys(COOH)-PEG-)_(n) chain.

Other synthetic polymers that can be used to conjugate haptens and/orprodrugs include N-(2-hydroxypropyl)methacrylamide (HMPA) copolymers,poly(styrene-co-maleic acid/anhydride (SMA), poly(divinylether maleicanhydride) (DIVEMA), polyethyleneimine, ethoxylated polyethylene-imine,starburst dendrimers and poly(N-vinylpyrrolidone) (PVP). As an example,DIVEMA polymer comprised of multiple anhydride units is reacted with alimited amount of SN-38 to produce a desired substitution ratio of drugon the polymer backbone. Remaining anhydride groups are opened underaqueous conditions to produce free carboxylate groups. A limited numberof the free carboxylate groups are activated using standardwater-soluble peptide coupling agents, (e.g.,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)), andcoupled to a recognition moiety bearing a free amino group. An exampleof the latter is histamine, to which antibodies have been raised in thepast.

The above exemplifications of polymer/drug conjugates embody the use ofSN-38, which is the active metabolite of the prodrug CPT-11(irinotecan). SN-38 has an aromatic hydroxyl group that was used in theabove descriptions to produce aryl esters susceptible to esterase-typeenzymes. Similarly the camptothecin analog topotecan, widely used inchemotherapy, has an available aromatic hydroxyl residue that can beused in a similar manner as described for SN-38, producingesterase-susceptible polymer-prodrugs. Water soluble derivatives ofcamptothecin are described in U.S. Pat. No. 4,943,579, incorporatedherein by reference. Conjugation of camptothecins to poly-(L-glutamicacid) has been described. See Singer et al., Annals of the N.Y. Acad.Sci., 922:136-150 (2000).

Doxorubicin also contains aromatic hydroxyl groups that can be coupledto carboxylate-containing polymeric carriers using acid-catalyzedreactions similar to those described for the camptothecin family.Similarly, doxorubicin analogs like daunomycin, epirubicin andidarubicin can be coupled in the same manner. Doxorubicin and otherdrugs with amino ‘chemical handles’ active enough for chemical couplingto polymeric carriers can be effectively coupled to carrier moleculesvia these free amino groups in a number of ways. Polymers bearing freecarboxylate groups can be activated in situ (EDC) and the activatedpolymers mixed with doxorubicin to directly attach the drug to theside-chains of the polymer via amide bonds. Amino-containing drugs canalso be coupled to amino-pendant polymers by mixing commerciallyavailable and cleavable cross-linking agents, such as ethyleneglycobis(succinimidylsuccinate) (EGS, Pierce Chemical Co., Rockford,Ill.) or bist2-(succinimido-oxycarbonyloxy)ethyl]sulfone (BSOCOES,Molecular Biosciences, Huntsville, Ala.), to cross-link the two aminesas two amides after reaction with the bis(succinimidyl) ester groups.This is advantageous as these groups remain susceptible to enzymaticcleavage. For example, (doxorubicin-EGS)_(n)-poly-lysine remainssusceptible to enzymatic cleavage of the diester groups in the EGSlinking chain by enzymes such as esterases. Doxorubicin also can beconjugated to a variety of peptides, for example,HyBnK(DTPA)YK(DTPA)-NH₂, using established procedures(HyBn=p-H₂NNHC₆H₄CO₂H). See Kaneko et al., J. Bioconjugate Chem., 2:133-141, 1991.

In one preferred embodiment, a therapeutic conjugate can be synthesizedwhich includes camptothecin, a derivative of camptothecin, ordoxorubicin coupled to a carrier comprising amine residues and achelating agent, such as DTPA, to form a DTPA-peptide-doxorubicinconjugate, wherein the DTPA forms the recognition moiety for apretargeted bsAb. Preferably, the carrier comprises a tyrosyl-lysinedipeptide, (e.g., Tyr-Lys(DTPA)-NH₂), and more preferably still itcomprises Lys(DTPA)-Tyr-Lys(DTPA)-NH₂. Doxorubicin phenyl hydrazoneconjugated to bis-DPTA containing peptides are particularly desirable ina therapeutic context.

Methotrexate also has an available amino group for coupling to activatedcarboxylate-containing polymers, in a similar manner to that describedfor doxorubicin. It also has two glutamyl carboxyl groups (alpha andgamma) that can be activated for coupling to amino-group containingpolymers. The free carboxylate groups of methotrexate can be activatedin situ (EDC) and the activated drug mixed with an amino-containingpolymer to directly attach the drug to the side-chains of the polymervia amide bonds. Excess unreacted or cross-reacted drug is separatedreadily from the polymer-drug conjugate using size-exclusion orion-exchange chromatography.

Maytansinoids and calicheamicins (such as esperamycin) contain mixed di-and tri-sulfide bonds that can be cleaved to generate species with asingle thiol useful for chemical manipulation. The thiomaytensinoid orthioespera-mycin is first reacted with a cross-linking agent such as amaleimido-peptide that is susceptible to cleavage by peptidases. TheC-terminus of the peptide is then activated and coupled to anamino-containing polymer such as polylysine.

Conjugation of Compounds to Lipids

The aforementioned compounds (e.g., targetable constructs and componentsthereof) and binding molecules (e.g., bsAbs) may be conjugated to: (1)lipids capable of delivering an effector (such as a drug); (2) moleculesthat can form a higher-ordered structure, (such as amphiphilic lipids orpolymers), which are capable of delivering an effector (such as a drug);and/or (3) higher-ordered structures capable of delivering an effector,(such as a micelle, liposome, polymeric structure, or nanoparticle). Theformation of liposomes, micelles, and emulsions is known in the art.(See, e.g., Wrobel et al., Biochimica et Biophysica Acta, 1235:296(1995); Lundberg et al., J. Pharm. Pharmacol., 51:1099-1105 (1999);Lundberg et al., Int. J. Pharm., 205:101-108 (2000); Lundberg, J. Pharm.Sci., 83:72-75 (1994); Xu et al., Molec. Cancer Ther., 1:337 -346(2002); Torchilin et al., Proc. Nat'l. Acad. Sci., 100:6039-6044 (2003);U.S. Pat. No. 5,565,215; U.S. Pat. No. 6,379,698; and U.S.2003/0082154). Nanoparticles or nanocapsules formed from polymers,silica, or metals, which are useful for drug delivery or imaging, havebeen described as well. (See, e.g., West et al., Applications ofNanotechnology to Biotechnology, 11:215-217 (2000); U.S. Pat. No.5,620,708; U.S. Pat. No. 5,702,727; and U.S. Pat. No. 6,530,944).

Where the targeting molecule is conjugated to a lipid, preferably thelipid is capable of forming an emulsion or a higher-ordered structuresuch as a micelle or liposome. For example, the lipid may be amphiphilic(e.g., a phospholipid). To facilitate conjugation to a targetableconstruct, the lipid may contain one or more groups capable of reactingwith the targetable construct such as nucleophilic carbons, (e.g., at adistal terminus). Polyethyleneglycol (PEG)-maleimide is a suitablelipid, wherevby the maleimide can react with free thiol groups presenton the targetable construct (e.g., on reduced cysteine residues).Maleimide groups may also be present on other carriers as describedherein for conjugating targetable constructs or binding molecules. Forexample, nanoparticles may contain maleimide groups for conjugating atargetable construct. In addition to maleimide groups, other groups forconjugating targetable constructs or binding molecules may includevinylsulfones as described in U.S. Pat. No. 6,306,393. Thelipid-conjugated, targetable constructs may form emulsions or liposomesthat can incorporate effector molecules as described herein (e.g.,hydrophobic drugs).

The conjugation of antibodies or binding molecules to lipids to form atargeted carrier for therapeutic or diagnostic agents has beendescribed. (See, e.g., Bendas, Biodrugs, 15:215-224 (2001); Xu et al.,Molec. Cancer Ther., 1:337-346 (2002); Torchilin et al., Proc. Nat'l.Acad. Sci., 100:6039-6044 (2003); Bally, et al., J. Liposome Res.,8:299-335 (1998); Lundberg, Int. J. Pharm., 109:73-81 (1994); Lundberg,J. Pharm. Pharmacol., 49:16-21 (1997); Lundberg, Anti-cancer DrugDesign, 13:453-461 (1998)). See also U.S. Pat. No. 6,306,393; U.S. Ser.No. 10/350,096; U.S. Ser. No. 09/590,284; U.S. Ser. No. 60/138,284,filed Jun. 9, 1999; and U.S. Ser. No. 60/478,830, filed Jun. 17, 2003.All these references are incorporated herein by reference. The samechemistry used to conjugate binding molecules (i.e., targetingmolecules) to lipids may be utilized to conjugate targetable constructsto lipids.

Preparation of Drug-Loaded Emulsions

Lipid-conjugated molecules can form emulsions or liposomes that includeeffectors such as drugs. The emulsions are composed of two major parts:(1) an oil core, (e.g., triglyceride); and (2) emulsifiers thatstabilize the oil core, (e.g., phospholipids). Triolein (TO), eggphosphatidylcholine (EPC), dipalmitoyl phosphatidylethanolamine (DPPE),cholesterol (CHOL), 8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS),polyoxyethylenesorbitan monooleate (sorbitan 80),methoxypolyethyleneglycol (PEG mean mol. wt 2000), oleoyl chloride,3(4,5-ditnethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) andDL-dithiotreitol (DTT) are obtained from commercial sources such asSigma Chemical Co. (St. Louis, Mo.). Poly(ethyleneglycol)-maleimide-N-hydroxy-succinimidyl ester (MAL-PEG₂₀₀₀-NHS) can bepurchased from Shearwater Polymers Europe (Enschede, The Netherlands).[³H]Cholesteryl oleoyl ether (COE) and [¹⁴C]dipalmitoylphosphatidylcholine are obtained from Amersham International plc(Amersham, UK). A PEG₂₀₀₀ derivative of dipalmitoylphosphatidylethanolamine (DPPE) with a maleimide group at the distalterminus of the PEG chain (DPPE-PEG-MAL) is synthesized by reacting 25mol NHS-PEG-MAL with 23 mol DPPE and 50 mol triethylamine in chloroformfor 6 h at 40° C. The product can be purified by preparative silica gelTLC.

Submicron lipid emulsions can be prepared as described in detailelsewhere. (See, Lundberg, J. Pharm. Sci., 83:72-75 (1994); Lundberg etal., Int. J. Pharm., 134:119-127 (1996); U.S. Ser. No. 60/478,830, filedJun. 17, 2003; and U.S. Pat. No. 6,306,393). The drug-loaded emulsionsinclude TO, EPC, polysorbate 80, DPPE-PEG₂₀₀₀-MAL, and an effector (suchas the drug FUdR-dO), at a ratio of 2:2:0.8:0.6:0.3 (w/w). Thecomponents are dispensed into vials from stock solutions and the solventis evaporated to dryness under reduced pressure. Phosphate-bufferedsaline (PBS) is added and the mixture is heated to 50° C.; vortex-mixedfor 30 s; and sonicated with a Branson probe sonicator for 2 min.

Drug loaded liposomes are composed of EPC, DPPE-PEG₂₀₀₀-MAL, FUdR-dO1:0.2:0.1 (w/w). A ratio of EPC, CHOL, DPPE-PEG₂₀₀₀-MAL 2:0.5:0.4 issuitable. Dried lipid films are hydrated in 25 mM HEPES and 140 mM NaClbuffer (pH 7.4), (containing 35 mM HPTS when appropriate); and aresubsequently subjected to five freezing-thawing cycles and sonicationfor 2 min with a Branson probe sonicator. The phospholipid concentrationare quantitated by incorporating [¹⁴C]DPPC.

Conjugation of Lipid Drug-Carriers to Targetable Constructs

Coupling of the aforementioned compounds (i.e., targetable constructs)or binding molecules to lipid drug-carriers can be performed by reactingthe maleimide (MAL) groups at the distal PEG termini on the surface of acarrier and a free thiol group, or other suitable group, on a targetableconstruct or binding molecule. Where the targetable construct or bindingmolecule contains disulfide groups, the disulfide groups can be reducedbefore the coupling reaction with 50 mM dithiotreitol for 1 h at 4° C.in 0.2 M tris buffer (pH 6.5) to provide free thiol groups. The reducedmolecule can be separated from excess dithiotreitol by use of SephadexG-25 spin-columns, equilibrated with 50 mM sodium acetate buffered 0.9%saline (pH 5.3). The conjugation can be performed in HEPES-bufferedsaline (pH 7.4) for 16 h at room temperature under argon. Excessmaleimide groups can be blocked with 2 mM 2-mercaptoethanol for 30 min,whereafter excess Ab and 2-mercaptoethanol can be removed on a SepharoseCL-4B column. The conjugated-liposomes can be collected near the voidvolume of the column, passed through a 0.22 μm sterile filter and storedat 4° C. The coupling efficiency can be estimated by use of afluorescein labeled targetable construct or binding molecule.

Compounds for Combined Therapeutic and Diagnostic Methods

In still other embodiments, the bi-specific antibody-directed deliveryof therapeutics or prodrug polymers to in vivo targets can be combinedwith bi-specific antibody delivery of radionuclides, such thatcombination chemotherapy and radioimmunotherapy is achieved. Eacheffector (e.g., nuclide and drug), may be conjugated to ornon-covalently associated with the targetable construct and administeredsimultaneously, or the nuclide can be given as part of a firsttargetable construct and the drug given in a later step as part of asecond targetable construct. In one embodiment, a peptide containing asingle prodrug and a single nuclide is constructed. For example, thetripeptide Ac-Glu-Gly-Lys-NH₂ can be used as a carrier portion of atargetable construct, whereby SN-38 is attached to the gamma glutamylcarboxyl group as an aryl ester, while a chelate is attached to theepsilon amino group as an amide, to produce the complexAc-Glu(SN-38)-Gly-Lys(chelate)-NH₂. The chelate can then be radiolabeledwith various metals for imaging and therapy purposes including ¹¹¹In,⁹⁰Y, ¹⁵³Sm, ¹⁷⁷Lu and ⁸⁹Zr. Because the metal-chelate complex canrepresent the recognized hapten on the targetable construct, theantibody can be designed to recognize and bind a selected metal-chelatecomplex at a sufficiently high affinity. Generally, this affinity (logK_(a)) is between 6-11. Polymeric peptides can be given as readily asthe more chemically defined lower MW reagent above, and are indeedpreferred. Also, triply substituted polymers can be used, such aspoly[Glu(Sn-38)₁₀-Lys(Y-90-chelate)_(n)(histamine-succinate)_(m), wheren and m are integers, such that the recognition agent is independent ofthe radioimmunotherapy agent. The prodrug can be activated bycarboxylesterases present at the tumor site or by carboxylesterasestargeted to the site using a second targetable construct.

Alternatively, a combination therapy can be achieved by administeringthe chemotherapy and radioimmunotherapy agents in separate steps. Forexample, a patient expressing CEA-tumors is first administered bsAb withat least one arm which specifically binds CEA and at least one other armwhich specifically binds the targetable construct whose hapten is acomplex (e.g., indium-DTPA or yttrium-DOTA). Later the patient istreated with a targetable construct comprising a conjugate (e.g.,indium-DTPA-beta-glucuronidase or yttrium-DOTA-beta-glucuronidase. Aftersufficient time for bsAb and enzyme localization and clearance, a secondtargetable construct, comprisingAc-Cys(Campto-COCH₂)-Lys(indium-DTPA)-Tyr-Lys(indium-DTPA)-NH₂ orAc-Glu(SN-38)-Gly-Lys(Y-90-DOTA)-NH₂, is given. The second targetableconstruct localizes to the tumor by virtue of bsAb at the tumor that arenot already bound to a first targetable construct. First targetableconstructs which are localized to the target site act on theAc-Cys(CPT)-Lys(indium-DTPA)-Tyr-Lys(indium-DTPA)-NH₂ orAc-Glu(SN-38)-Gly-Lys(Y-90-DOTA)-NH₂ to liberate CPT or SN-38.Localization of both the prodrug and its respective enzyme to the targetsite enhances the production of active drug by ensuring that the enzymeis not substrate limited. This embodiment constitutes a markedimprovement of current prodrug methodologies currently practiced in theart.

Another advantage of administering the prodrug-polymer in a later step,after the nuclide has been delivered as part of a previously giventargetable construct, is that the synergistic effects of radiation anddrug therapy can be manipulated and, therefore, maximized. It ishypothesized that tumors become more ‘leaky’ after RAIT due to radiationdamage. This can allow a polymer-prodrug to enter a tumor morecompletely and deeply. This results in improved chemotherapy.

Alternatively, the RAIT therapy agent can be attached to the bsAb ratherthan to the targetable construct. For example, an anti-CEA×anti-DTPAbsAb conjugated to Y-90-DOTA can be administered first to a patient withCEA-expressing tumors. In this instance, advantage is taken of theselectivity of certain anti-chelate mabs in that an anti-indium-DTPAantibody do not bind to a yttrium-DOTA chelate. After theY-90-DOTA-anti-CEA×anti-indium-DTPA has maximized at the tumor andsubstantially cleared non-target tissue, a conjugate ofindium-DTPA-glucuronidase is injected and localized specifically to theCEA tumor sites. The patient is then injected with a polymer-prodrugsuch as poly(Glu)(SN-38)₁₀. The latter is cleaved selectively at thetumor to active monomeric SN-38, successfully combining chemotherapywith the previously administered RAIT.

Antibodies

Bi-specific antibodies or antibody fragments can be used in the presentmethod, with at least one binding site specific to an antigen at atarget site and at least one other binding site specific to the enzymecomponent of the antibody-enzyme conjugate. Such an antibody can bindthe enzyme prior to injection, thereby obviating the need to covalentlyconjugate the enzyme to the antibody. Alternatively, the antibody can beinjected and localized at the target site and, after non-targetedantibody has substantially cleared from the circulatory system of themammal, an enzyme can be injected in an amount and by a route whichenables a sufficient amount of the enzyme to reach a localized antibodyor antibody fragment and bind to it to form the antibody-enzymeconjugate in situ.

The methods disclosed herein also contemplate the use of multivalenttarget binding proteins which have at least three different targetbinding sites as described in Patent Appl. Ser. No. 60/220,782 filedJul. 25, 2000. Multivalent target binding proteins have been made bycross-linking several Fab-like fragments via chemical linkers. See U.S.Pat. Nos. 5,262,524; 5,091,542 and Landsdorp et al., Euro. J. Immunol.16: 679-83 (1986). Multivalent target binding proteins also have beenmade by covalently linking several single chain Fv molecules (scFv) toform a single polypeptide. See U.S. Pat. No. 5,892,020. A multivalenttarget binding protein which is basically an aggregate of scFv moleculeshas been disclosed in U.S. Pat. Nos. 6,025,165 and 5,837,242. Atrivalent target binding protein comprising three scFv molecules hasbeen described in Krott et al., Protein Engineering 10(4): 423-433(1997).

Clearing Agents

A clearing agent can be used which is given between doses of the bsAband the targetable construct. It has been discovered that a clearingagent of novel mechanistic action can be used with the methods describedherein, namely a glycosylated anti-idiotypic Fab′ fragment targetedagainst the disease targeting arm(s) of the bsAb. Anti-CEA (MN-14Ab)×anti-peptide bsAb is given and allowed to accrete in disease targetsto its maximum extent. To clear residual bsAb, an anti-idiotypic Ab toMN-14, termed WI2, is given, preferably as a glycosylated Fab′ fragment.The clearing agent binds to the bsAb in a monovalent manner, while itsappended glycosyl residues direct the entire complex to the liver, whererapid metabolism takes place. Then the therapeutic or diagnostic agentwhich is associated with the targetable construct is given to thesubject. The WI2 Ab to the MN-14 arm of the bsAb has a high affinity andthe clearance mechanism differs from other disclosed mechanisms (seeGoodwin et al., ibid), as it does not involve cross-linking, because theWI2-Fab′ is a monovalent moiety. Clearing agents and uses thereof aredescribed in U.S. Pat. No. 6,667,024; U.S. Pat. No. 6,468,530; U.S. Pat.No. 6,387,350; U.S. Pat. No. 6,096,289; U.S. Pat. No. 5,922,302; U.S.Pat. No. 5,736,119; U.S. Pat. No. 5,698,405; U.S. Pat. No. 5,698,178;U.S. Pat. No. 5,686,578; and U.S. Pat. No. 5,525,338; all of which areincorporated herein by reference in their entireties.

Kits

The compounds may be packaged as a kit suitable for use in treating oridentifying diseased tissues in a patient by performing the methodsdisclosed herein. Minimally, the kit includes one or more of thecompounds herein (e.g., as a targetable construct or targetablemolecule). The kit can also include one or more binding molecules (e.g.,antibodies or fragments thereof as targeting molecules) and/or one ormore clearing agents. The kit can also include instruments whichfacilitate identifying or treating diseased tissue. Examples include,but are not limited to application devices, such as syringes. The kitcan also include solutions required for identifying or treating diseasedtissue. The kit can also include instructions and/or labels withinstructions.

Methods for Raising Antibodies

Abs to peptide backbones and/or haptens are generated by well-knownmethods for Ab production. For example, an immunogen can be injectedinto an immunocompetent animal. The immunogen may include a peptideconjugated to KLH, (e.g., (peptide)_(n)-KLH, wherein KLH is keyholelimpet hemocyanin, and n=1-30, in complete Freund's adjuvant). Theprimary injection may be followed by two subsequent injections of thesame immunogen suspended in incomplete Freund's adjuvant, and theseinjections may be followed by a subsequent i.v. boost of antigen (i.e.,peptide). Three days after the i.v. boost of antigen, spleen cells areharvested and fused with Sp2/0-Ag14 myeloma cells. Culture supernatantsof the resulting clones are then analyzed for anti-peptide reactivityusing a direct-binding ELISA. Fine specificity mapping of the generatedAbs can be analyzed by using peptide fragments of the originalantigen/peptide. These fragments can be prepared readily using anautomated peptide synthesizer. For Ab production, enzyme-deficienthybridomas are isolated to enable selection of fused cell lines. Thistechnique also can be used to raise antibodies to one or more of thechelates comprising the linker, e.g., In(III)-DTPA chelates. Monoclonalmouse antibodies to an In(III)-di-DTPA are known (Barbet '395 supra).

The antibodies used in the present compounds and methods are specific toa variety of cell surface or intracellular tumor-associated antigens asmarker substances. These markers may be substances produced by the tumoror may be substances which accumulate at a tumor site, on tumor cellsurfaces or within tumor cells, whether in the cytoplasm, the nucleus orin various organelles or sub-cellular structures. Among suchtumor-associated markers are those disclosed by Herberman,“Immunodiagnosis of Cancer”, in Fleisher ed., “The Clinical Biochemistryof Cancer”, page 347 (American Association of Clinical Chemists, 1979)and in U.S. Pat. Nos. 4,150,149; 4,361,544; and 4,444,744. See also U.S.Pat. No. 5,965,132, to Thorpe et al., U.S. Pat. No. 6,004,554, to Thorpeet al., U.S. Pat. No. 6,071,491, to Epstein et al., U.S. Pat. No.6,017,514, to Epstein et al., U.S. Pat. No. 5,882,626, to Epstein etal., U.S. Pat. No. 5,019,368, to Epstein et al., and U.S. Pat. No.6,342,221, to Thorpe et al., and U.S. patent application Ser. Nos.09/337,756; 09/382,186; 09/823,746; and 10/150,654 all of which areincorporated herein by reference.

Tumor-associated markers have been categorized by Herberman, supra, in anumber of categories including oncofetal antigens, placental antigens,oncogenic or tumor virus associated antigens, tissue associatedantigens, organ associated antigens, ectopic hormones and normalantigens or variants thereof. Occasionally, a sub-unit of atumor-associated marker is advantageously used to raise antibodieshaving higher tumor-specificity, e.g., the beta-subunit of humanchorionic gonadotropin (HCG) or the gamma region of carcinoembryonicantigen (CEA), which stimulate the production of antibodies having agreatly reduced cross-reactivity to non-tumor substances as disclosed inU.S. Pat. Nos. 4,361,644 and 4,444,744. Markers of tumor vasculature(e.g., VEGF), of tumor necrosis (Epstein patents), of membrane receptors(e.g., folate receptor, EGFR), of transmembrane antigens (e.g., PSMA),and of oncogene products can also serve as suitable tumor-associatedtargets for antibodies or antibody fragments. Markers of normal cellconstituents which are expressed copiously on tumor cells, such asB-cell complex antigens (e.g., CD19, CD20, CD21, CD22, CD23, and HLA-DRon B-cell malignancies), as well as cytokines expressed by certain tumorcells (e.g., IL-2 receptor in T-cell malignancies) are also suitabletargets for the antibodies and antibody fragments as used herein. Otherwell-known tumor associated antigens that can be targeted by theantibodies and antibody fragments used herein include, but are notlimited to, CEA, CSAp, TAG-72, MUC-1, MUC-2, MUC-3, MUC-4, EGP-1, EGP-2,BrE3, PAM-4, KC-4, A3, KS-1, PSMA, PSA, tenascin, T101, S100, MAGE,HLA-DR, CD19, CD20, CD22, CD30, CD74, IFG, ILG-1, and IL-6.

Preferred bi-specific antibodies are those which incorporate the Fv ofMAb Mu-9 and the Fv of MAb 734, the Fv of MAb MN-14 and the Fv of MAb734, the Fv of MAb RS-7 and the Fv of MAb 734, the Fv of MAb Mu-9 andthe Fv of MAb 679, the Fv of MAb RS-7 and the Fv of MAb 679, or the Fvof MAb MN-14 and the Fv of MAb 679, and their human, chimerized orhumanized counterparts. The monoclonal antibody MN-14, as well as itschimerized and humanized counterparts, are disclosed in U.S. Pat. No.5,874,540. Also preferred are bi-specific antibodies which incorporateone or more of the CDRs of Mu-9 or 679. Particularly suitablebi-specifica antibodies may include LL2×734, LL2×679, PAM4×734,PAM4×679, LL1×734, and LL1×679. The antibody can also be a fusionprotein or a bi-specific antibody that incorporates a Class-III anti-CEAantibody and the Fv of 679. Class-III antibodies, including Class-IIIanti-CEA are discussed in detail in U.S. Pat. No. 4,818,709.

The present invention is further illustrated by, though in no waylimited to, the following examples.

EXAMPLES Example 1 Synthesis of 20-O-chloroacetyl camptothecin

20-O-chloroacetyl camptothecin has been described. See U.S. Pat. No.4,943,579. Camptothecin (1.0 gm) was dissolved in 40 mL of CHCl₃.Chloroacetic anhydride (1.2 eq), pyridine (1.0 eq) and DMAP (0.1 eq) wasadded to this mixture which was then refluxed for two hours. After noobservable change in the reaction mixture, additional chloroaceticanhydride (1.2 eq) and pyridine (1.0 eq) was subsequently added and themixture was refluxed for an additional 2 hrs. HPLC showed the reactiontaking place. An additional amount of chloroacetic anhydride (2.1 eq)and pyridine (4.3 eq) was added and the reaction mixture was refluxedfor another 2 hrs. HPLC showed the reaction to be complete. Mixture wasworked up by washing with 65 mL of H₂O, then 0.1N NaOH solution, thenanother 65 mL of H₂O. Organic layer was dried with Na₂SO₄ then filteredand finally removed under reduced pressure. Yellow precipitate formed.HPLC showed one product. ESMS results show MH⁺: 425. Final yield afterdrying: 1.178 g (2.772×10⁻³ mol, 96.5%).

Example 2 Synthesis of IMP 274,Ac-Cys(Campto-COCH₂)-Lys(DTPA)-Tyr-Lys(DTPA)-NH₂

The IMP 274 peptide (see FIG. 1) was synthesized using a protocolsimilar to that described by Karacay et. al. Bioconjugate Chem.11:842-854 (2000). IMP 222, (Ac-Cys-Lys(DTPA)-Tyr-Lys(DTPA)-NH₂) (38.1mg) and 11.7 mg (1.0 eq) of 20-O-chloroacetyl camptothecin were eachdissolved separately in 150 μL DMF. The two quantities were combined andstirred. Pyridine (100 μL) was added and the reaction vessel was purgedwith Argon and sealed with parafilm. Little change was noted by HPLCafter 1.5 hrs. DIEA (50 mL) was then added to the reaction mixture. HPLCshows the reaction to have gone to completion after 2 hrs. The reactionmixture was purified using a prep column and fractions were sent foranalysis by ESMS (MH⁺: 1721). Four fractions show pure product with RTof ˜7.1 minutes. Final yield: 22.2 mg (1.290×10⁻⁵ mol, 46.9%).Similarly, chemistry well known in the art can be used to synthesisderivatives of IMP 274 as shown in FIGS. 2-4.

Example 3 IMP 274 Labeling Kit

Labeling kits were made by dissolving citric acid (0.414 gm), HPCD(5.0074 gm), in 80 mL of DI H₂O. This mixture was then adjusted to pH4.25 after which IMP 274 (0.0021 gm) was added. The volume was QS to 100mL with DI H₂O and 1 mL aliquots of this solution, filtered through a0.22 μm filter, were added to lyophilization vials which were thenfrozen, lyophilized, and stoppered under vacuum.

Example 4 IMP 274 Peptide Labeling

A IMP 274 In-111 labeling kit was dissolved in 0.5 mL DI water. 110 μLof the solution was removed and placed in an acid washed Eppendorf tube.1.8 mCi of the In-111 (preferably from Perkin Elmer) was added to theEppendorf tube. The solution was allowed to incubate at room temperaturefor 20 min then 250 μL of the 1.0×10⁻⁴ M In(III) solution (0.1 M NaOAcpH4.5) was added. The solution containing the cold indium was incubatedat room temperature for 30 min. An aliquot, 140 μL, was removed anddiluted with PBS or saline to 7.0 mL in a serum stoppered vial.

Example 5 Stability of In₂-IMP 274 Kits in an In-Vitro TestingFormulation Buffer

Lyophilized kits containing 1 mg of IMP 274 were prepared for in-vitrotesting. The kits contain 2-hydroxypropyl-β-cyclodextrin (an excipientand solubilizer), a buffer, and cold Indium to form a complex with theDTPA moieties. To test stability after a repeated freeze-thaw cycle, thekits were thawed; aliquots were withdrawn and examined by reverse phaseHPLC and size exclusion HPLC (without dilution or manipulation). Thesolutions were examined using a Waters 4.6×250 mm X Terra RP C₁₈ 5 μmcolumn. The UV was monitored at 220 nm. The HPLC conditions were asfollows: flow rate of 1 mL/min, linear gradient 100% A (0.1% TFA inwater) to 100% B (90% CH₃CN, 10% water, 0.1% TFA) over 30 min. Thepeptide demonstrated stability in the formulation buffer. See FIGS. 7and 8.

Example 6 IMP 274 Labeling and Stability Studies

IMP 274 was labeled with ¹¹¹In. After being labeled, this protein wastested for stability in human and nude mouse serum over a period of 24hrs. The studies show that the peptide does undergo stability changes inthe presence of both human serum (t_(1/2)=4 hrs.) and mouse serum(t_(1/2)=18 hrs.). IMP 274 was also tested for binding with thehumanized antibody, m734×hMN14. Studies were analyzed both on thereverse phase and the size exclusion HPLC systems.

Example 7 IMP 274 Labeling: Mouse and Human Labeling, CompletedSeparately

¹¹¹InCl₃ (31 μL and 21 μL) was added respectively to 500 μL of DI H₂O.These quantities were added to the prepared vial of IMP 274 and allowedto sit for approximately 20 minutes. An additional 900 μL of cold InAcetate Buffer (1.0×10⁻⁴M, 0.5M NaOAc, pH 6.5) was added to each andwere allowed to sit for an additional 45 minutes. The total volume foreach vial was 1431 μL and 1421 μL with a molar quantity of 1.220×10⁻⁸moles of IMP 274 yielding a concentration of 8.526×10⁻⁶M for the mouseserum study and 8.586×10⁻⁶M for the human serum study.

Example 8 IMP 274 Mouse and Human Serum Stability

One labeled peptide mixture, 50 μL, was combined with 450 μL fresh mouseand another with human serum. These were vortexed and placed under aconstant temperature of 37° C. Samples were analyzed, at various timepoints, by HPLC for stability between 0 hr, and 23 hrs. The radiometricchromatograms show that the labeled peptide changed over time. (FIGS. 9and 10). The dilutions of 50 μL of peptide in 500 μL of solution changedthe concentration of both mixtures to 8.526×10⁻⁷M for the mouseexperiment and 8.586×10⁻⁷M human serum experiment.

Example 9 Addition of Antibody and Mouse Serum to IMP 274

¹¹¹In-IMP 274 (10 μL) was added to 3 μL of the antibody(antibody/peptide ratio of ˜22:1) and 290 μL of 0.9% Saline. The mixturewas vortexed and analyzed by size exclusion HPLC. (FIG. 11).

Example 10 Addition of Antibody and Human Serum to IMP 274

¹¹¹In-IMP 274 (16 μL) was added to 10 μL of antibody (antibody/peptideratio of ˜23:1) and 24 μL 0.9% Saline. The mixture was vortexed andanalyzed by size exclusion HPLC. (FIG. 12).

Example 11 Synthesis of IMP 156 (Ac-Phe-Lys(DTPA)-Tyr-Lys-DTPA-NH₂)

The peptide on the resin was synthesized by reacting the resin with sixequivalents of amino acid per coupling. The activating agents werediisopropylcarbodiimide and N-hydroxybenzotriazole. The couplings wererun at room temperature overnight. The resin (2.109 gAc-Phe-Lys(Aloc)-Tyr(But)-Lys(Aloc)NH-Sieber Amide Resin (˜7×10⁻⁴ mol))was washed with 2×40 mL CH₂Cl₂. Tributyltin hydride, 5 mL was added tothe resin. Piperidine, 2 mL was mixed with 1 mL of acetic acid, themixture became hot and crystals formed. The crystals were dissolved in40 mL CH₂Cl₂ and mixed with 0.729 g Pd[P(Ph)₃]₄. This solution was addedto the resin mixture and mixed at room temperature for 1.5 hr. Thecleavage solution was drained from the resin. The resin was then treatedwith a second one hour treatment with fresh Aloc cleavage reagents. Theresin was washed with 40 mL portions of 3×CH₂Cl₂, 2×50 mL portions of25% piperidine in DMF, 40 mL portions of NMP, IPA, NMP, IPA, IPA, and4×NMP. DTPA tetra-t-butyl ester, 3.679 g (5.95×10⁻³ mol) was dissolvedin 20 mL NMP and mixed with 1 mL diisopropylcarbodiimide and 0.991 gN-hydroxybenzotriazole monohydrate. This solution was incubated at roomtemperature for 10 min and then added to the resin. The DTPA was reactedwith the resin for 15 hr at room temperature. The resin was then washedwith 40 mL portions of NMP, IPA, NMP, IPA, IPA, (the resin was ninhydrinnegative) 4×NMP, 4×CH₂Cl₂ and then dried under a stream of nitrogen. Thepeptide was cleaved from the resin by a 3 hr treatment with a solutioncontaining 14 mL TFA, 0.5 mL triisopropylsilane, and 0.5 mL anisole. Thecrude peptide was precipitated in ether, collected by centrifugation anddried in a vacuum oven at room temperature. The crude peptide wasresuspended in TFA for 1.5 hr to finish cleaving the protecting groupsfrom the peptide. The peptide was purified by reverse phase HPLC using0.1% TFA buffers to afford 0.54 g of pure product after lyophilization(ESMS MH⁺1377).

Example 12 Synthesis of IMP 222 (Ac-Cys-Lys(DTPA)-Tyr-Lys(DTPA)-NH₂

Fmoc-Lys(Aloc)-Tyr(But)-Lys(Aloc)-NH-Sieber Amide Resin (5.148 g,˜1.0×10⁻² mol) was added to a column, rinsed with 50 mL NMP, and thenswelled by addition of a second 50 mL portion of NMP. N₂ gas was addedand bubbled through the resin for ˜30 minutes (i.e., the column wasbubbled). The solution was removed and 40 mL 25% Piperidine/NMP wasadded. The column was bubbled for 4 minutes and the solution wasremoved. A second 40 mL portion of 25% Piperidine/NMP was added. Thecolumn was bubbled for an additional 15 minutes and the solution wasremoved. The resin was rinsed with 50 mL portions of NMP, IPA, NMP, IPA,and then 4×NMP. Fmoc-Cys(Trt)OH (5.860 g), and N-hydroxybenzotriazole(1.535 g) were both dissolved in ˜35 mL NMP. Diisopropylcarbodiimide(1.6 mL) was added. After the reagents were dissolved, the solution wasadded to the resin and the column was bubbled using N₂ gas for ˜18 hrs.The solution was removed and the resin was rinsed with 50 mL portions ofNMP, IPA, NMP, IPA, and then 4×NMP. A 25% Piperidine/NMP solution wasadded. The column was again bubbled with N₂ gas for 4 minutes and thesolution was removed. This was repeated again for 15 minutes with the25% Piperidine/NMP solution and the solution was removed. The resin wasrinsed with 50 mL portions of NMP, IPA, NMP, IPA, and then 4×NMP. AceticAnhydride (4.8 mL) was added to 40 mL of NMP and thenDiisopropylethylamine (8.9 mL) was added. This solution was added to theresin and the column was bubbled with N₂ gas for ˜2 hrs. The resin wasrinsed with 50 mL portions of NMP, IPA, NMP, IPA, and then 4×NMP. Theresin was then rinsed with 2×50 mL CH₂Cl₂. Tributyltin hydride (5 mL)was added to the resin. A previously mixed solution ofTetrakis(Triphenylphosphine)Palladium(0) (1.095 g), Glacial acetic acid(2 mL), Piperidine (4 mL) and ˜55 mL of CH₂Cl₂ was also added to theresin. The column was bubbled with N₂ gas for ˜2 hrs. and the solutionwas removed. The procedure was repeated with Tributyltin hydride (5 mL),Tetrakis(Triphenylphosphine)Palladium(0) (1.001 g), Glacial acetic acid(2 mL), Piperidine (4 mL) and ˜55 mL of CH₂Cl₂. The column was bubbledwith N₂ gas for ˜1 hr. and the solution was removed. The resin wasrinsed with 50 mL portions of NMP, IPA, NMP, IPA, and then 4×NMP. Tetrat-butyl ester DTPA (7.087 g) was dissolved in ˜25 mL NMP. To thissolution was added N-hydroxybenzotriazole (1.715 g) anddiisopropylcarbodiimide (1.8 mL). The column was bubbled with N₂ gas for˜18 hrs and the solution was removed. The resin was rinsed with 50 mLportions of NMP, IPA, NMP, IPA, and then 4×NMP. This was followed by3×50 mL rinses of CH₂Cl₂. The resin was dried by slow purge of N₂ gasfor ˜30 minutes. The peptide was cleaved from the resin and deprotectedby addition of premixed solution consisting of 60 mL Trifluoroaceticacid, 2 mL Anisole, and 2 mL Triisopropylsilane. The column was bubbledwith N₂ gas for ˜2 hrs, the solution was removed, and the supernatantwas collected. The resin was rinsed with an additional amount of 25 mLTrifluoroacetic acid, which was collected as well. The supernatant waspoured into 50 mL polyethylene centrifuge tubes (˜10 mL per tube). Thepeptide was precipitated out of solution by addition of 40 mL Diethylether to each followed by vortexing and centrifuging for approximately 5minutes. The supernatant was decanted and the procedure was repeatedtwice more. The remaining crude peptide was dried under vacuumovernight. The crude peptide was redissolved with approximately 10 mLtrifluoroacetic acid and monitored by HPLC due to incomplete cleavage.The precipitation procedure using diethyl ether was repeated after ˜2hrs. All the crude dried peptide was combined and dissolved in 8 mL ofdeionized water. The peptide solution was loaded onto a Waters RCM®.Preperative Column and, using mobile phases A (0.1% TFA in DI H₂O) and B(0.1% TFA in 90% Acetonitrile/10% DI H₂O), purified using a gradient of100%/0% to 70%/30% over 80 minutes at 65 mL min⁻¹. Fractions #7,8,9, &10contained pure material by analytical HPLC. The fractions were frozenand lyophilized to yield a total of 521.4 mg of pure material. Sampleswere sent for ESMS analysis which showed MH⁺: 1333 and [M−H]⁻: 1331 foreach fraction.

Example 13 Synthesis of Bromoacetyl Doxorubicin

Doxorubicin hydrochloride, 0.9993 g (1.72×10⁻³ mol) was dissolved in 10mL DMF and mixed with 0.5 mL pyridine. The solution was cooled in an icebath. Bromo acetyl bromide, 160 μL (1.84×10⁻³ mol), was added to thereaction mixture. Diisopropylethyl amine, 0.6 mL was added after 3.5 hr.HPLC analysis showed mostly starting material so the starting materialwas precipitated by mixing with ether. The precipitate was washed with2×30 mL, portions of ether and dried in a vacuum oven at roomtemperature. The doxorubicin was redissolved in 10 mL DMF.Diisopropylethylamine, 1 mL, was added followed by the addition of 250μL (2.87×10⁻³ mol) of additional bromo acetyl bromide. The solution wasmixed for 17 min and precipitated with 60 mL ether. The red solidprecipitate was washed with three additional 60 mL portions of ether.The red solid precipitate was resuspended in 10 mL CH₃CN andprecipitated by pouring into 50 mL of ether to obtain a red powder. Thered powder was collected by centrifugation and was again washed with3×60 mL portions of ether. The crude product was then purified by flashchromatography by dissolving the crude product in CHCl₃ and placing onflash silica ¾ full in a 150 mL sintered glass funnel. The silica waseluted with 200 mL portions of ether, chloroform, 4×95:5chloroform/methanol and 2×90:10 chloroform/methanol. The product was inthe second (0.1390 g) and third (0.2816 g) 95:5 chloroform/methanolfractions.

Example 14 Synthesis of IMP 225

The bromoacetyl doxorubicin (0.0714 g, 1.08×10⁻⁴ mol) was mixed with0.1333 g (1.00×10⁻⁴ mol) IMP 222 and dissolved in 1.0 mL DMF. Potassiumbicarbonate, 0.4544 g was suspended in 1 mL H₂O and then added to theDMF solution, which warmed slightly. The reaction mixture was incubatedat room temperature overnight. The reaction mixture was poured into 30mL of ether in a 50 mL centrifuge tube. The contents of the tube weremixed and the ether layer was decanted. The ether wash was repeated witha second 30 mL portion of ether. The solution was then acidified with 1M HCl and purified in two portions by HPLC on a Waters 19×300 mm PrepNova-Pak HRC18 6 μm 60 Å column. The gradient started at 100% 0.1% TFAin water (Buffer A) flowing at 25 mL/min and, using a linear gradient,it went to 50:50 Buffer A:B (buffer B being 90% CH₃CN 0.1% TFA, 10% H₂O)over 80 min. The fractions containing the desired product were collectedand lyophilized to afford 0.0895 g (47% yield) of the desired product(ESMS MH⁺ 1916). See FIG. 5 for chemical structure of IMP 225.

Example 15 Synthesis of IMP 294 (In₂-Labeled IMP 225)

A 0.1 M citric acid buffer was prepared by dissolving 0.386 g of citricacid in 15 mL H₂O. The buffer solution was adjusted to pH 3.60 by theaddition of 1 M NaOH and the solution was diluted to 20 mL. The peptide,0.293 g (1.53×10⁻⁵ mol, 100 mol %) was mixed with 0.0128 g (5.79×10−5mol, 378 mol %) InCl₃ and dissolved in 5 mL of the citrate buffer. Thereaction solution was incubated at room temperature overnight and thenpurified by HPLC on a Waters 19×300 mm Prep Nova-Pak HRC18 6 μm 60 Åcolumn. The gradient started at 90% Buffer A (defined above) and 10%Buffer B (defined above) flowing at 25 mL/min and, using a lineargradient, it went to 60:40 Buffer A:B over 80 min. The fractionscontaining the desired product were collected and lyophilized to afford0.0160 g (49% yield) of the desired product (ESMS MH⁻ 2318).

Example 16 Synthesis of IMP 295 (In₂-Labeled IMP 156)

The peptide, IMP 156, 0.1299 g (9.43×10⁻⁵ mol, 100 mol %) was mixed with0.0740 g (3.34×10⁻⁴ mol, 355 mol %) and dissolved in 5 mL of the 0.1 M,pH 3.6 citrate buffer. The reaction solution was incubated at roomtemperature for ˜4 hr then purified by HPLC on a Waters 19×300 mm PrepNova-Pak HRC18 6 μm 60 Å column. The gradient started at 100% Buffer A(defined above) flowing at 25 mL/min and, using a linear gradient, itwent to 50:50 Buffer A:B over 80 min. The fractions containing thedesired product were collected and lyophilized to afford 0.1095 g (73%yield) of the desired product (ESMS MH⁻ 1598).

Example 17 Stability of IMP 294 and IMP 295 in PBS at 25° C.

A phosphate buffered saline (PBS) solution was prepared by mixing 2.535g Na₂HPO₄, 0.450 g NaH₂PO₄.H₂O, 4.391 g NaCl and diluting to 500 mL withH2O. A stock solution of IMP 294 was prepared by dissolving 0.0011 g ofthe peptide in 5.00 mL of PBS. A 2.0 mL aliquot of the stock solutionwas removed and mixed with 4.9 mL of PBS to provide a 3×10⁻⁵ M solutionof the IMP 294 (In₂ IMP 225) in PBS. A stock solution of IMP 295 wasprepared by dissolving 0.0011 g of the peptide in 5.00 mL of PBS. A 2.0mL aliquot of the stock solution was removed and mixed with 7.2 mL ofPBS to provide a 3×10⁻⁵ M solution of the IMP 295 (In₂ IMP 156) in PBS.The samples were incubated in the auto-injector of the Waters AllianceHPLC at 25° C. The samples were analyzed by reverse phase HPLC using aWaters Xterra™ RP₁₈ 5 μm 4.6×250 mm column, part number W10891R 015,which was heated at 25° C. The flow rate for the column was 1 mL/min andthe eluent was monitored at 220 nm with a PDA detector. A lineargradient was used starting at 100% Buffer A going to 100% Buffer B over30 min. Injections, (100 μL) were made daily for one week. See FIGS. 13Aand B.

Example 18 Synthesis of IMP 224

An amount of 0.0596 g of the phenyl hydrazine containing peptide IMP 221(H₂N—NH—C₆H₄—CO-Lys(DTPA)-Tyr-Lys(DTPA)-NH₂ MH⁺ 1322, made by Fmoc SPPS)was mixed with 0.0245 g of Doxorubicin hydrochloride in 3 mL of DMF. Thereaction solution was allowed to react at room temperature in the dark.After 4 hours an additional 0.0263 g of IMP 221 was added and thereaction continued overnight. The entire reaction mixture was thenpurified by HPLC on a Waters Nova-Pak (3-40×100 mm segments, 6 μm, 60 Å)prep column eluting with a gradient of 80:20 to 60:40 Buffer A:B over 40min (Buffer A=0.3% NH₄OAc, Buffer B=0.3% NH₄OAc in 90% CH₃CN). Thefractions containing product were combined and lyophilized to afford0.0453 g of the desired product, which was confirmed by ESMS MH⁺ 1847.See FIG. 6 for chemical structure of IMP 224.

Example 19 IMP 224 Kit Formulation

The peptide of Example 14 was formulated into kits for In-111 labeling.A solution was prepared which contained 5.014 g2-hydroxypropyl-β-cyclodextrin, and 0.598 g citric acid in 85 mL. Thesolution was adjusted to pH 4.20 by the addition of 1 M NaOH and dilutedwith water to 100 mL. An amount of 0.0010 g of the peptide IMP 224 wasdissolved in 100 mL of the buffer, and 1 mL aliquots were sterilefiltered through a 0.22 μm Millex GV filter into 2 mL lyophilizationvials which were immediately frozen and lyophilized.

Example 20 In-111 Labeling of IMP 224 Kits

The In-111 was dissolved in 0.5 mL water and injected into thelyophilized kit. The kit solution was incubated at room temperature for10 min then 0.5 mL of a pH 7.2 buffer which contained 0.5 M NaOAc and2.56×10⁻⁵ M cold indium was added.

Example 21 In-Vitro Stability of IMP 224 Kits

An IMP 224 kit was labeled as described with 2.52 mCi of In-111.Aliquots (0.15 mL, 370 μCi) were withdrawn and mixed with 0.9 mL 0.5 Mcitrate buffer pH 4.0, 0.9 mL 0.5 M citrate buffer pH 5.0, and 0.9 mL0.5 M phosphate buffer pH 7.5. The stability of the labeled peptide wasfollowed by reverse phase HPLC. HPLC Conditions: Waters Radial-Pak C-18Nova-Pak 8×100 mm, Flow Rate 3 mL/min, Gradient: 100% A=0.3% NH₄OAc to100% B=90% CH₃CN, 0.3% NH₄OAc over 10 min. The stability results areshown in Table 1.

TABLE 1 In-Vitro Stability of In/In-111 IMP 224 Kit pH 4.0 pH 5.0 pH 7.5% % % % Intact Intact Intact Intact Pep- Pep- Pep- Pep- Time tide Timetide Time tide Time tide 0 100 24 min 100  0 100 24 min 100  2 hr 100* 2hr 100* 21 hr  89 19 hr 25 21 hr  89 19 hr 25 *Some peptide decomposedbut was not included in the calculation of the areas of the peaks

Example 22 Biodistribution of In-111-Labeled IMP 274 and I-125-LabeledhRS-7×MAb 734 in CALU-3 Tumor Bearing Nude Mice and Pretargeting withhRS-7×MAb 734

Seventy nude mice were implanted with CALU-3 cells. These mice were usedto look at the possibility of doing pretargeting with EGP-1 expressingtumors. Tumor binding and in-vivo clearance of the bispecific antibodyI-125 hRS-7×734 was assayed using time points of 2, 4, 24 and 48 hr. Inaddition, we performed a pretargeting experiment with I-125 hRS-7×734.For the pretargeting experiment the peptide In-111/In IMP 274 wasinjected at 16 and 24 hr after the injection of the bispecific antibodyhRS-7×734. The animals in the pretargeting study were sacrificed at 3and 24 hr after the injection of the peptide. A third group of animalsreceived only the In-111/In IMP 274 peptide. These animals weresacrificed at 1, 3, 6 and 24 hr post injection of the peptide.Typically, five mice were used for each time point. The followingtissues were weighed and counted: Tumor, Liver, Spleen, Kidney, Lung,Blood, Stomach, Small Intestine, Large Intestine, Heart and Urine.

Antibody Clearing Group: At least 35 animals were injected with 100 μLof a solution containing the bispecific antibody I-125 hRS-7×734 (5 μCi,15 μg, 1.5×10⁻¹⁰ mol). The 20 animals in the antibody clearance groupwere split into five groups of five animals each and sacrificed at 2, 4,24 and 48 hr postinjection.

Pretargeting Group: The 30 animals in the pretargeting group were splitinto six groups of approximately five animals each. Approximately 24hours later, the I-125 labeled bispecific antibody was injected intoanother 10 animals, and the peptide 100 μL (10 μCi, 1.5×10⁻¹¹ mol) wasinjected into those 10 animals at 4 hr postinjection of the antibody.The animals were sacrificed at 3 and 24 hr after the injection of thepeptide. Approximately 24 hours later, the peptide, 100 μL (10 μCi,1.5×10⁻¹¹ mol), was injected into 10 animals at 24 hr postinjection ofthe antibody. The animals were sacrificed at 3 and 24 hr after theinjection of the peptide. Approximately 48 hours later, 10 animals wereinjected with 100 μL of the peptide. Five animals per time point weresacrificed at 3 hr and 24 hr post injection of the peptide.

Peptide Alone Group: Approximately 24 hours after the start of thestudy, the peptide 100 μL (10 μCi, 1.5×10⁻¹¹ mol) was injected into 15animals. The animals were split into three groups and sacrificed at 1,3, 6 and 24 hr post injection of the peptide. The results of the studyare summarized in Tables 2-9.

TABLE 2 In-111/IMP 274 Biodistribution in CALU-3 Tumor Bearing NudeMice, % ID/g Tissue 1 Hr 3 Hr 6 Hr 24 Hr Tumor 0.32 ± 0.11 0.14 ± 0.020.11 ± 0.02 0.13 ± 0.02 Liver 0.11 ± 0.02 0.10 ± 0.01 0.10 ± 0.02 0.10 ±0.02 Spleen 0.11 ± 0.02 0.10 ± 0.01 0.09 ± 0.01 0.09 ± 0.02 Kidney 2.24± 0.66 1.93 ± 0.18 1.59 ± 0.18 1.05 ± 0.18 Lung 0.22 ± 0.04 0.12 ± 0.050.11 ± 0.04 0.06 ± 0.01 Blood 0.25 ± 0.06 0.12 ± 0.02 0.09 ± 0.02 0.03 ±0.00 Stomach 0.09 ± 0.05 0.12 ± 0.02 0.03 ± 0.01 0.03 ± 0.01 Sm. Int.0.25 ± 0.13 0.21 ± 0.10 0.06 ± 0.01 0.08 ± 0.02 Large Int. 0.13 ± 0.140.62 ± 0.38 0.22 ± 0.05 0.06 ± 0.01 Heart 0.10 ± 0.02 0.07 ± 0.01 0.06 ±0.01 0.04 ± 0.01 Urine 269 ± 366 7.28 ± 10.9 0.82 ± 0.56 0.20 ± 0.10

TABLE 3 I-125-labeled hRS-7 x 734 Biodistribution in CALU-3 TumorBearing Nude Mice, % ID/g Tissue 2 Hr 4 Hr 24 Hr 48 Hr Tumor 2.80 ± 0.813.98 ± 1.71 5.10 ± 2.20 1.84 ± 0.40 Liver 2.80 ± 0.70 2.94 ± 1.08 0.67 ±0.14 0.19 ± 0.01 Spleen 2.37 ± 1.08 2.95 ± 0.86 0.63 ± 0.20 0.15 ± 0.05Kidney 5.73 ± 1.36 5.37 ± 1.42 0.70 ± 0.16 0.13 ± 0.01 Lung 3.46 ± 1.732.81 ± 1.20 0.71 ± 0.19 0.09 ± 0.02 Blood 13.2 ± 2.49 9.69 ± 2.28 1.53 ±0.22 0.14 ± 0.02 Stomach 3.41 ± 0.58 6.59 ± 2.36 1.83 ± 1.21 0.17 ± 0.05Sm. Int. 1.61 ± 0.40 1.47 ± 0.45 0.39 ± 0.11 0.05 ± 0.00 Large Int. 0.82± 0.22 1.02 ± 0.28 0.36 ± 0.11 0.06 ± 0.01 Heart 2.74 ± 0.99 2.92 ± 0.940.52 ± 0.07 0.05 ± 0.01 Urine 4.05 ± 1.96 3.13 ± 2.36 4.05 ± 3.54 1.40 ±0.72

TABLE 4 In-111-labeled IMP 274 Biodistribution in CALU-3 Tumor BearingNude Mice Pretargeted with hRS-7 x m73. Peptide Injected 4, 24, & 48 hrPost bsAb Injection, 10:1 bsAb/peptide Ratio. % ID/g Determined 3 HrPost Injection of the Peptide. Tissue 4 hr Post bsAb 24 hr Post bsAb 48hr Post bsAb Tumor 2.88 ± 1.54 10.3 ± 1.89 4.58 ± 1.02 Liver 3.73 ± 1.420.92 ± 0.26 0.14 ± 0.04 Spleen 2.95 ± 0.71 0.82 ± 0.30 0.14 ± 0.02Kidney 5.15 ± 0.53 2.61 ± 0.25 1.32 ± 0.50 Lungs 2.63 ± 0.67 0.79 ± 0.140.17 ± 0.05 Blood 11.4 ± 3.60 2.06 ± 0.59 0.23 ± 0.03 Stomach 1.22 ±0.23 0.42 ± 0.21 0.10 ± 0.11 Sm Int 2.31 ± 0.46 0.52 ± 0.25 0.11 ± 0.05Large Int 2.54 ± 0.96 0.56 ± 0.19 0.86 ± 0.48 Heart 2.54 ± 0.71 0.67 ±0.16 0.09 ± 0.01 Urine 39.8 ± 33.5 22.4 ± 25.0 7.55 ± 4.89

TABLE 5 In-111-labeled IMP 274 Biodistribution in CALU-3 Tumor BearingNude Mice Pretargeted with hRS-7 x m734. Peptide injected 4, 24 & 48 hrPost bsAb Injection, 10:1 bsAb/peptide Ratio. Tumor/nontumor (T/NT)Ratios Determined 3 Hr Post Injection of the Peptide. Tissue 4 hr PostbsAb 24 hr Post bsAb 48 hr Post bsAb Tumor — — — Liver 0.76 ± 0.43 11.8± 4.68 34.4 ± 8.97 Spleen 0.92 ± 0.44 14.0 ± 6.58 33.7 ± 3.78 Kidney0.54 ± 0.28 3.88 ± 0.85 3.86 ± 1.36 Lungs 1.07 ± 0.59 13.1 ± 3.55 28.9 ±6.19 Blood 0.24 ± 0.12 5.29 ± 2.09 19.5 ± 3.46 Stomach 2.30 ± 1.21 33.2± 24.7 95.4 ± 73.9 Sm Int 1.29 ± 0.76 25.4 ± 16.9 47.5 ± 21.1 Large Int1.40 ± 1.09 19.9 ± 7.80 8.23 ± 6.86 Heart 1.08 ± 0.53 16.0 ± 5.76 51.4 ±9.73 Urine 0.20 ± 0.22 10.6 ± 22.0 2.06 ± 3.32

TABLE 6 In-111-labeled IMP 274 Biodistribution in CALU-3 Tumor BearingNude Mice Pretargeted with hRS-7 x m73. Peptide Injected 4, 24, & 48 hrPost bsAb Injection, 10:1 bsAb/peptide Ratio. % ID/g Determined 24 HrPost Injection of the Peptide. Tissue 4 hr Post bsAb 24 hr Post bsAb 48hr Post bsAb Tumor 5.26 ± 2.76 9.66 ± 0.50 4.79 ± 1.11 Liver 4.95 ± 2.510.90 ± 0.20 0.17 ± 0.03 Spleen 4.95 ± 2.62 1.19 ± 0.32 0.16 ± 0.04Kidney 4.75 ± 1.55 2.22 ± 0.68 1.21 ± 0.29 Lungs 0.93 ± 0.49 0.36 ± 0.090.11 ± 0.04 Blood 0.60 ± 0.22 0.31 ± 0.06 0.07 ± 0.02 Stomach 0.39 ±0.24 0.09 ± 0.03 0.05 ± 0.05 Sm Int 0.64 ± 0.22 0.21 ± 0.04 0.08 ± 0.01Large Int 0.76 ± 0.34 0.18 ± 0.02 0.09 ± 0.04 Heart 2.03 ± 0.99 0.42 ±0.11 0.07 ± 0.01 Urine 5.68 ± 3.42 1.00 ± 1.02 0.56 ± 0.53

TABLE 7 In-111-labeled IMP 274 Biodistribution in CALU-3 Tumor BearingNude Mice Pretargeted with hRS-7 x m734. Peptide injected 4, 24 & 48 hrPost bsAb Injection, 10:1 bsAb/peptide Ratio. T/NT Ratios Determined 24Hr Post Injection of the Peptide. Tissue 4 hr Post bsAb 24 hr Post bsAb48 hr Post bsAb Tumor — — — Liver 1.06 ± 0.14 11.2 ± 2.49 29.2 ± 12.5Spleen 1.10 ± 0.19 8.54 ± 2.18 30.0 ± 7.89 Kidney 1.07 ± 0.31 4.72 ±1.63 4.07 ± 1.01 Lungs 5.90 ± 1.47 27.9 ± 6.69 47.5 ± 12.4 Blood 8.53 ±1.80 32.5 ± 6.72 70.6 ± 24.7 Stomach 15.4 ± 5.88  117 ± 47.7  137 ± 77.1Sm Int 7.90 ± 1.89 47.3 ± 9.61 59.4 ± 16.1 Large Int 6.81 ± 0.99 55.4 ±8.46 64.7 ± 28.2 Heart 2.57 ± 0.26 24.3 ± 6.19 74.1 ± 19.9 Urine 0.99 ±0.17 25.9 ± 28.2 120 ± 216

TABLE 8 In-111-labeled IMP 274 Biodistribution in CALU-3 Tumor BearingNude Mice Pretargeted with 250 mg hRS-7 x m734. Peptide Injected 48 hrPost bsAb Injection, 136:1 bsAb/peptide Ratio. % ID/g and T/NT RatiosDetermined 3 & 24 hr Post Injection of the Peptide. Tissue 3 hr % ID/g 3hr T/NT 24 hr % ID/g 48 hr T/NT Tumor 10.9 ± 3.14 — 8.48 ± 2.77 — Liver3.18 ± 1.21 3.80 ± 1.53 2.23 ± 1.27 4.51 ± 1.91 Spleen 2.56 ± 0.92 4.62± 1.59 1.25 ± 0.26 6.98 ± 2.31 Kidney 4.52 ± 0.78 2.42 ± 0.49 1.91 ±0.41 4.55 ± 1.42 Lung 3.49 ± 1.36 3.59 ± 1.72 0.66 ± 0.21 13.6 ± 4.51Blood 12.8 ± 4.53 0.96 ± 0.44 1.13 ± 0.51 8.49 ± 3.72 Stomach 1.00 ±0.27 12.0 ± 6.36 0.19 ± 0.07 48.0 ± 19.1 Sm. Int. 0.94 ± 0.34 13.2 ±6.34 0.26 ± 0.05 34.4 ± 13.5 Large Int. 0.60 ± 0.11 18.5 ± 5.14 0.25 ±0.06 34.6 ± 11.2 Heart 2.95 ± 1.26 4.41 ± 2.35 0.64 ± 0.30 15.4 ± 7.59Urine 33.2 ± 42.7 — 2.84 ± 0.71 3.14 ± 1.21

TABLE 9 I-125-labeled hRS-7 x 734 Biodistribution in CALU-3 TumorBearing Nude Mice Pretargeted 48 hr Prior to Peptide Injection with 250mg hRS-7 x m734, 136:1 bsAb/peptide Ratio. % ID/g and T/NT RatiosDetermined 3 & 24 hr Post Injection of the Peptide. Tissue 3 hr % ID/g 3hr T/NT 24 hr % ID/g 48 hr T/NT Tumor 10.9 ± 3.14 — 8.48 ± 2.77 — Liver0.19 ± 0.06 11.5 ± 4.82 0.10 ± 0.02 9.10 ± 1.61 Spleen 0.22 ± 0.07 9.79± 4.40 0.10 ± 0.06 12.2 ± 6.42 Kidney 0.29 ± 0.14 7.85 ± 3.09 0.13 ±0.05 7.93 ± 2.80 Lung 0.27 ± 0.10 8.04 ± 2.69 0.09 ± 0.01 10.3 ± 1.84Blood 0.18 ± 0.06 11.3 ± 2.40 0.05 ± 0.03 21.0 ± 6.42 Stomach 0.45 ±0.29 5.36 ± 1.93 0.16 ± 0.06 6.52 ± 2.95 Sm. Int. 0.08 ± 0.02 24.4 ±5.01 0.04 ± 0.01 26.5 ± 7.48 Large Int. 0.12 ± 0.03 17.7 ± 6.69 0.05 ±0.01 21.0 ± 6.42 Heart 0.08 ± 0.02 24.5 ± 3.43 0.03 ± 0.01 35.4 ± 12.3Urine 2.02 ± 2.61 7.46 ± 8.98 0.64 ± 0.22 1.51 ± 0.36

Example 23 Biodistribution of In-111-Labeled IMP 274 in GW-39 TumorBearing Nude Mice and in GW-39 Tumor Bearing Nude Mice Pretargeting withhMN-14×m734

Biodistribution of In-111-Labeled IMP 274 was determined in GW-39 TumorBearing Nude Mice and is summarized in Table 10. Biodistribution ofIn-111-Labeled IMP 274 in GW-39 Tumor Bearing Nude Mice pretargeted withhMN-14×m734 is shown in Tables 11.

TABLE 10 In-111-Labeled IMP 274 Biodistribution in GW-39 Tumor BearingNude Mice. ID/g Determined at 0.5 hr, 1 hr, 4 hr, and 24 hr PostInjection of the Peptide. Tissue 0.5 hr 1 hr 4 hr 24 hr Tumor 1.44 ±0.37 0.91 ± 0.07 0.23 ± 0.05 0.12 ± 0.01 Liver 0.35 ± 0.07 0.29 ± 0.100.18 ± 0.03 0.20 ± 0.03 Spleen 0.34 ± 0.11 0.20 ± 0.04 0.08 ± 0.04 0.16± 0.01 Kidney 4.48 ± 0.83 2.75 ± 0.56 1.77 ± 0.32 1.65 ± 0.32 Lung 0.75± 0.21 0.39 ± 0.20 0.10 ± 0.01 0.07 ± 0.01 Blood 1.12 ± 0.24 0.60 ± 0.090.18 ± 0.10 0.06 ± 0.01 Stomach 0.22 ± 0.07 0.13 ± 0.02 0.09 ± 0.04 0.13± 0.12 Sm. Int. 0.35 ± 0.10 0.33 ± 0.06 0.54 ± 0.39 0.18 ± 0.08 LargeInt. 0.31 ± 0.17 0.13 ± 0.06 0.17 ± 0.08 0.14 ± 0.06 Heart 0.41 ± 0.070.51 ± 0.52 0.07 ± 0.01 0.04 ± 0.02 Urine 1034 ± 563  1050 ± 549  4.35 ±5.62 0.21 ± 0.05

TABLE 11 In-111-Labeled IMP 274 Biodistribution in GW-39 Tumor BearingNude Mice Pretargeted 24 hr Prior to Peptide Injection with hMN-14 xm734. ID/g Determined at 0.5 hr, 1 hr, 4 hr, and 24 hr Post Injection ofthe Peptide. Tissue 0.5 hr 1 hr 4 hr 24 hr Tumor 5.59 ± 2.01 5.96 ± 2.756.79 ± 3.33 6.16 ± 3.87 Liver 0.72 ± 0.17 0.43 ± 0.14 0.66 ± 0.11 0.43 ±0.06 Spleen 0.67 ± 0.14 0.62 ± 0.15 0.71 ± 0.18 0.49 ± 0.17 Kidney 5.53± 1.42 2.90 ± 0.54 5.74 ± 1.11 2.46 ± 0.48 Lungs 1.65 ± 0.62 1.10 ± 0.291.39 ± 0.36 0.83 ± 0.22 Blood 3.40 ± 1.13 2.09 ± 0.72 3.18 ± 1.08 2.06 ±0.75 Stomach 0.40 ± 0.11 0.38 ± 0.11 0.43 ± 0.16 0.23 ± 0.08 Sm Int.0.60 ± 0.16 0.56 ± 0.10 0.60 ± 0.11 0.56 ± 0.07 Large Int. 0.28 ± 0.040.29 ± 0.03 0.24 ± 0.04 0.17 ± 0.04 Heart 0.96 ± 0.40 0.67 ± 0.16 0.78 ±0.31 0.48 ± 0.15 Urine 1560 ± 105  227 ± 279  549 ± 22.2  170 ± 67.2

Example 24 Biodistribution of In-111-Labeled IMP 225 in GW-39 TumorBearing Nude Mice and in GW-39 Tumor Bearing Nude Mice Pretargeting withhMN-14×m734

Biodistribution of In-111-Labeled IMP 225 was determined in GW-39 TumorBearing Nude Mice and is summarized in Table 12. Biodistribution ofIn-111-Labeled IMP 225 in GW-39 Tumor Bearing Nude Mice pretargeted withhMN-14×m734 is shown in Table 13.

TABLE 12 In-111-Labeled IMP 225 Biodistribution in GW-39 Tumor BearingNude Mice. ID/g Determined at 0.5 hr, 1 hr, 4 hr, and 24 hr PostInjection of the Peptide. Tissue 0.5 hr 1 hr 4 hr 24 hr Tumor 1.62 ±0.45 1.03 ± 0.26 0.21 ± 0.03 0.10 ± 0.01 Liver 0.39 ± 0.05 0.26 ± 0.120.19 ± 0.05 0.14 ± 0.02 Spleen 0.21 ± 0.06 0.69 ± 1.19 0.11 ± 0.02 0.08± 0.02 Kidney 6.86 ± 1.41 4.18 ± 1.06 2.66 ± 0.69 1.21 ± 0.39 Lung 0.72± 0.21 0.54 ± 0.50 0.14 ± 0.04 0.04 ± 0.01 Blood 1.28 ± 0.23 0.62 ± 0.350.18 ± 0.04 0.04 ± 0.01 Stomach 0.27 ± 0.05 0.55 ± 0.37 0.14 ± 0.10 0.06± 0.03 Sm. Int. 0.39 ± 0.07 0.43 ± 0.11 0.16 ± 0.06 0.10 ± 0.02 LargeInt. 0.32 ± 0.10 0.16 ± 0.07 0.47 ± 0.21 0.16 ± 0.04 Heart 0.30 ± 0.070.49 ± 0.68 0.06 ± 0.01 0.03 ± 0.01 Urine 1500 ± 263  552 ± 493 3.12 ±2.23 0.21 ± 0.03

TABLE 13 In-111-Labeled IMP 225 Biodistribution in GW-39 Tumor BearingNude Mice Pretargeted 24 hr Prior to Peptide Injection with hMN-14 xm734. ID/g Determined at 0.5 hr, 1 hr, 4 hr, and 24 hr Post Injection ofthe Peptide. Tissue 0.5 hr 1 hr 4 hr 24 hr Tumor 7.84 ± 3.66 9.00 ± 2.085.71 ± 2.47 5.21 ± 1.27 Liver 0.99 ± 0.04 0.93 ± 0.25 0.61 ± 0.14 0.51 ±0.10 Spleen 0.67 ± 0.10 0.43 ± 0.09 0.36 ± 0.19 0.34 ± 0.06 Kidney 10.6± 4.53 6.01 ± 2.70 2.84 ± 0.38 2.17 ± 0.80 Lungs 1.42 ± 0.33 0.89 ± 0.220.59 ± 0.49 0.16 ± 0.04 Blood 3.92 ± 0.20 2.98 ± 0.63 0.93 ± 0.26 0.23 ±0.05 Stomach 0.85 ± 0.20 0.62 ± 0.31 0.19 ± 0.05 0.11 ± 0.02 Sm Int.0.70 ± 0.31 0.49 ± 0.12 0.27 ± 0.07 0.21 ± 0.08 Large Int. 0.59 ± 0.240.29 ± 0.07 0.44 ± 0.13 0.21 ± 0.07 Heart 0.80 ± 0.09 0.58 ± 0.09 0.24 ±0.10 0.16 ± 0.04 Urine 920 ± 322 1082 ± 541  6.44 2.12 ± 0.79

Example 25 In-Vivo Biodistribution of IMP 224 in BALB/c Mice

Kits were reconstituted with 400 μCi In-111 in 0.5 mL water. The In-111kit solution was incubated at room temperature for 10 min and thendiluted with 1.5 mL of the cold indium containing pH 7.2, 0.5 M acetatebuffer. The labeled peptide was analyzed by ITLC in saturated NaCl. Theloose In-111 was at the top 20% of the ITLC strip.

Each mouse was injected with 100 μL (20 μCi) of the In-111 labeledpeptide. The animals were anesthetized and sacrificed at 30 minutes, 1hours, 2 hours, 4 hours, and 24 hours using three mice per time point.Blood, muscle, liver, lungs, kidneys, spleen, large intestine, smallintestine, stomach, urine, and tail were collected and counted. Theresults of the biodistribution study are shown in Table 14.

TABLE 14 Biodistribution in BALB/c mice % ID/g of IMP 224 (Dox =N—NH—C₆H₄—CO-Lys(DTPA)-Tyr- Lys(DTPA)-NH₂MH⁺ 1847 radiolabeled withIn-111 and saturated with cold indium. Tissue 30 min 1 hr 2 hr 4 hr 24hr Liver 0.57 ± 0.04 0.31 ± 0.03 0.17 ± 0.03 0.17 ± 0.01 0.13 ± 0.02Spleen 0.57 ± 0.18 0.27 ± 0.06 0.12 ± 0.01 0.11 ± 0.01 0.07 ± 0.00Kidney 8.45 ± 1.79 5.36 ± 1.01 3.75 ± 0.52 4.03 ± 0.45 2.12 ± 0.17 Lungs1.61 ± 0.34 0.99 ± 0.26 0.25 ± 0.02 0.17 ± 0.02 0.09 ± 0.02 Blood 1.44 ±0.28 0.54 ± 0.12 0.12 ± 0.01 0.10 ± 0.01 0.02 ± 0.00 Stomach 0.61 ± 0.070.15 ± 0.07 0.05 ± 0.01 0.06 ± 0.02 0.04 ± 0.02 Small Int. 0.72 ± 0.080.37 ± 0.19 0.09 ± 0.01 0.09 ± 0.03 0.05 ± 0.01 Large Int. 0.59 ± 0.430.18 ± 0.04 0.38 ± 0.15 0.30 ± 0.06 0.08 ± 0.03 Muscle 0.51 ± 0.19 0.21± 0.08 0.03 ± 0.02 0.02 ± 0.00 0.01 ± 0.00 Urine 1553 1400 ± 421  19.11.72 ± 0.67 0.42 ± 0.18 Tail 3.66 ± 0.43 1.90 ± 0.09 0.46 ± 0.09 0.24 ±0.03 0.58 ± 0.22

Example 26 In-Vivo Stability and Clearance of IMP 224

Kits were reconstituted with 4 mCi In-111 in 0.5 mL water. The In-111kit was incubated at room temperature for 10 min and then diluted with0.5 mL of the cold indium containing 0.5 M pH 7.2 acetate buffer. Thelabeled peptide was analyzed by ITLC in saturated NaCl. The loose In-111was at the top 20% of the ITLC strip.

Each mouse was injected with 100 μL (400 μCi) of the In-111 labeledpeptide. The animals were anesthetized and sacrificed at 30 min and 1 hrusing two animals per time point. The serum and urine samples werecollected, stored on ice, and sent on ice as soon as possible for HPLCanalysis. The HPLC (by size exclusion chromatography) of the urinesamples showed that the In-111 labeled peptide could still bind to theantibody. The reverse phase HPLC analysis showed that the radiolabeledpeptide was excreted intact in the urine. The amount of activityremaining in the serum was too low to be analyzed by reverse phase HPLCdue to the poor sensitivity of the detector. Doxorubicin has ˜95%hepatobiliary clearance. Thus, by attaching the bis DTPA peptide in ahydrolyzable manner, the biodistribution of the drug is altered to give˜100% renal excretion. This renders the drug far less toxic because allof the nontargeted drug is rapidly excreted intact. Clearance resultsare shown in Table 15.

TABLE 15 Activity Recovered in The Urine and Serum 30 min 1 hr TissueAnimal #1 Animal #2 Animal #1 Animal #2 Urine  220 μCi 133 μCi 41.1 μCi 273 μCi Serum 1.92 μCi 3.64 μCi 1.21 μCi 1.27 μCi

Example 27 Pretargeting Experiments with IMP 224 and IMP 225

A lyophilized kit of IMP 224 containing 10 micrograms of peptide wasused. The kit was lyophilized in 2 mL vials and reconstituted with 1 mLsterile water. A 0.5 mL aliquot was removed and mixed with 1.0 mCiIn-111. The In-111 kit solution was incubated at room temperature for 10minutes then 0.1 mL was removed and diluted with 1.9 mL of the coldindium containing acetate buffer BM 8-12 in a sterile vial. The labeledpeptide was analyzed by ITLC in saturated NaCl. The loose In-111 was atthe top 20% of the ITLC strip.

Female nude mice (Taconic NCRNU, 3-4 weeks old) with GW 39 tumorxenografts were used for the pretargeting experiments. Tumors were0.3-0.8 g. Each animal was injected with 100 microliters (5 Ci, 15 g,1.5×10⁻¹⁰ mol) of the I-125 labeled antibody F6×734-F(ab′)₂.

Seventy two hours later, each mouse was injected with 100 L (10 Ci) ofthe In-111 labeled peptide. The animals were anesthetized and sacrificedat 1 hour, 4 hours and 24 hours using five mice per time point. Tumor,blood, muscle, liver, lungs, kidneys, spleen, large intestine, smallintestine, stomach, urine and tail were collected and counted.

The experiment was repeated with a lyophilized kit of IMP 225NH₂-Lys(DTPA)-Tyr-Lys(DTPA)-Cys(Dox-COCH₂)-Ac (SEQ ID NO: 4) MNa⁺ 1938),containing 11 micrograms of peptide. The biodistribution results aresummarized in Tables 16-18.

TABLE 16 Biodistribution of In-111-IMP-224 in nude mice bearing GW-39tumor xenografts, previously given F6 × 734-F(ab′)₂ 72 h earlier. Datain % ID/g tissue. n = 5. 1 h 4 h 24 h Tissue I-125 In-111 I-125 In-111I-125 In-111 GW- 10.0 ± 10.3 ± 9.8 ± 2.6 11.0 ± 8.8 ± 1.2 9.7 ± 1.1 391.5  1.7  2.0 Liver 0.1 ± 0.0 0.4 ± 0.1 0.1 ± 0.0  0.3 ± 0.0 0.1 ± 0.00.3 ± 0.0 Spleen 0.1 ± 0.0 0.4 ± 0.1 0.1 ± 0.0  0.2 ± 0.0 0.1 ± 0.0 0.2± 0.0 Kidney 0.3 ± 0.1 3.5 ± 0.6 0.2 ± 0.0  2.8 ± 0.3 0.2 ± 0.0 1.9 ±0.2 Lungs 0.2 ± 0.0 0.8 ± 0.2 0.2 ± 0.0  0.4 ± 0.0 0.2 ± 0.0 0.1 ± 0.0Blood 0.4 ± 0.1 1.8 ± 0.6 0.4 ± 0.1  0.9 ± 0.2 0.4 ± 0.0 0.2 ± 0.0 Stom-0.5 ± 0.2 0.8 ± 1.3 0.5 ± 0.2  0.1 ± 0.0 0.7 ± 0.2 0.1 ± 0.0 ach Small0.1 ± 0.0 0.5 ± 0.4 0.1 ± 0.0  0.2 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 Int. Large0.1 ± 0.0 0.1 ± 0.0 0.1 ± 0.0  0.3 ± 0.1 0.1 ± 0.1 0.1 ± 0.1 Int. Muscle0.0 ± 0.0 0.3 ± 0.2 0.0 ± 0.0  0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 Urine 1.1 ±2.0  168 ± 1.8 ± 0.6 31.8 ± 0.9 ± 0.2 1.2 ± 0.2 106   31   Tail 0.1 ±0.0 1.1 ± 0.2 0.1 ± 0.0  0.4 ± 0.1 0.2 ± 0.0 0.2 ± 0.0

TABLE 17 Biodistribution of In-111-IMP-224 in nude mice bearing GW-39tumor xenografts, previously given F6 × 734-F(ab′)₂ 72 h earlier. Datain tumor-to-normal organ ratios. n = 5. 1 h 4 h 24 h Tissue I-125 In-111I-125 In-111 I-125 In-111 GW- 1 1 1 1 1 1 39 Liver 85.4 ± 24.0 ± 81.8 ±35.4 ± 61.1 ± 31.6 ± 25    5.9 25    6.9  8.5  5.8 Spleen 81.0 ± 28.7 ±74.5 ± 44.7 ± 60.8 ± 47.0 ± 34    8.7 25   10    8.6  2.2 Kid- 39.7 ± 3.0 ± 57.1 ±  3.9 ± 39.6 ±  5.0 ± ney  9.4  0.5 14    0.5  4.8  0.5Lungs 51.2 ± 13.4 ± 50.7 ± 30.1 ± 50.3 ± 69.0 ± 10    2.7 10    4.9 10   9.4 Blood 25.2 ±  6.1 ± 22.9 ± 12.8 ± 21.8 ± 41.8 ±  8.3  2.5 7   2.0 4.2  6.3 Stom- 21.0 ± 48.7 ± 22.1 ±  128 ± 14.9 ±  147 ± ach  6.7 37  7  46    6.0 39   Small  137 ± 31.9 ±  128 ± 51.6 ±  102 ±  110 ± Int.41   18   37   14    3.7 13   Large  136 ± 87.1 ±  130 ± 45.6 ±  113 ±92.4 ± Int. 32   35   39   19   12   38   Mus-  209 ± 38.6 ± 1396 ±  727 ±  233 ±  283 ± cle 86   13   797   42   46   Urine 11.0 ±  0.3 ± 6.3 ± 0.71 ±  9.8 ±  8.3 ± 23    0.5  4.2  0.6  1.9  1.3 Tail 72.7 ± 9.4 ± 73.6 ± 26.4 ± 53.9 ± 55.9 ± 20    2.8 20    5.2 10    5.7

TABLE 18 Biodistribution of In-111-IMP-225 in nude mice bearing GW-39tumor xenografts, previously given F6 × 734-F(ab′)₂ 72 h earlier. Datain % ID/g tissue. n = 5. 1 h 4 h 24 h Tissue I-125 In-111 I-125 In-111I-125 In-111 GW- 6.2 ± 5.9 14.6 ± 10.5 ± 16.5 ± 8.3 ± 3.0 10.1 ± 39 143.8 4.8 2.3 Liver 0.1 ± 0.1 0.4 ± 0.2 0.2 ± 0.0 0.4 ± 0.1 0.1 ± 0.0 0.3± 0.1 Spleen 0.5 ± 0.7 1.6 ± 2.4 0.2 ± 0.1 0.4 ± 0.1 0.1 ± 0.0 0.4 ± 0.1Kid- 0.3 ± 0.1 3.8 ± 0.9 0.3 ± 0.1 3.8 ± 0.4 0.2 ± 0.1 1.7 ± 0.3 neyLungs 0.3 ± 0.1 0.8 ± 0.4 0.3 ± 0.0 0.6 ± 0.1 0.2 ± 0.1 0.2 ± 0.1 Blood0.5 ± 0.1 2.0 ± 0.4 0.8 ± 0.4 1.3 ± 0.2 0.3 ± 0.1 0.4 ± 0.2 Stom- 0.1 ±0.2 1.1 ± 0.9 0.8 ± 0.4 0.4 ± 0.2 0.3 ± 0.0 0.1 ± 0.0 ach Small 0.1 ±0.0 0.4 ± 0.1 0.1 ± 0.0 0.3 ± 0.2 0.1 ± 0.0 0.1 ± 0.0 Int. Large 0.1 ±0.0 0.2 ± 0.0 0.1 ± 0.0 0.3 ± 0.1 0.1 ± 0.0 0.1 ± 0.0 Int. Mus- 0.0 ±0.0 0.3 ± 0.2 0.1 ± 0.0 0.2 ± 0.0 0.0 ± 0.0 0.1 ± 0.0 cle Urine 2.8 ±3.4  110 ± 2.0 ± 1.0 13.5 ± 0.3 ± 0.3 0.7 ± 0.4 40 6.4 Tail 0.4 ± 0.21.2 ± 0.1 0.2 ± 0.0 0.8 ± 0.2 0.1 ± 0.1 0.5 ± 0.7

Example 28 Pretargeting in SCID Mice Inoculated with Daudi Cells

SCID mice were inoculated with Daudi (Burkitt's lymphoma) cells toproduce disseminating disease. One group of mice received four i.p.injections of LL2×734 administered on days 1, 3, 7, and 9. This wasfollowed by four i.p. injections of IMP-225 on days 2, 4, 8, and 10. Acontrol group (no IMP-225) received IMP-225 alone on days 2, 4, 8, and10. See FIG. 14A. Mice were observed daily for signs of paralysis as theendpoint of survival. Median percent survival was calculated andanalyzed using Kaplan-Meier plots (log-rank analysis). See FIG. 14B.

Example 29 Synthesis of DTPA

DTPA may be synthesized as outlined in the schematics shown in FIGS. 15and 16.

Three Step Method.

a. Synthesis of N-(2-((5-dibenzosuberyl)amino)ethyl)-1,2-ethanediamine,3: Diethylenetriamine 1, 350 mL, was poured into a 1000 ml three neckflask, which had been flushed with nitrogen. The solution was cooled inan ice/salt bath to 3° C. The protecting group precursor5-chlorodibenzosuberane 2 (15.017 g, 6.57×10⁻² mol) was slowly added byspoonfuls to the reaction mixture over a 15 min period under a positivepressure of nitrogen. The reaction was magnetically stirred and allowedto warm slowly to room temperature over 18 hr. The reaction was thencooled in the ice bath and 350 mL of water was slowly added (keeping thetemperature below 50° C.). The reaction mixture was extracted with 4×100mL CH₂Cl₂. The organic layers were combined and washed with 2×100 mLH₂O. The organic extracts were then dried over Na₂SO₄, filtered andconcentrated on the rotary evaporator to provide 19.258 g (99% yield) ofthe yellow oily product. ESMS MH⁺ 296.

b. Synthesis ofN,N,N′,N″-Tetra((tert-butoxy-carbonyl)methyl)-N″-(2-((5-dibenzosuberyl)amino)ethyl)-1,2-ethanediamine5: The crude N-(2-((5-dibenzosuberyl)amino)ethyl)-1,2-ethanediamine 3,53 g (1.8×10⁻¹ mol) was dissolved in 90 mL acetonitrile and placed undernitrogen. Diisopropylethyl amine, 71 mL (5.49×10⁻¹ mol, 836 M %) wasadded and the solution was cooled in an ice bath. Tert-Butylbromoacetate 4, 42 mL (2.48×10⁻¹ mol, 446 M %) was added dropwise, andthe reaction was allowed to warm slowly as it stirred overnight undernitrogen. The following day an additional 15 mL (4.06×10⁻² mol, 62 M %)was added. The reaction was stirred overnight at room temperature. Thereaction mixture then was concentrated on the rotary evaporator. Thecrude product was mixed with 200 mL ethyl acetate and extracted with2×100 mL and 2×50 ml saturated sodium bicarbonate. The organic solutionwas dried over Na₂SO₄, filtered, and concentrated on the rotaryevaporator to obtain 56 g of the crude product as an amber oil.

c. Synthesis of 1-tert-Butyl hydroxy3,6,9-Tris((tert-butoxycarbonyl)methyl)-3,6,9-triazaundecanedioic Acid7: The crudeN,N,N′,N″-Tetra((tert-butoxy-carbonyl)methyl)-N″-(2-((5-dibenzosuberyl)amino)ethyl)-1,2-ethanediamine5 was mixed with 36.541 g (3.97×10⁻¹ mol, 604 mol %) of glyoxylic acidmonohydrate 6 and dissolved in 50 ml methanol. The Parr bottle wasflushed with nitrogen and the catalyst 1.721 g (10% palladium on carbon)was added. After two days, an additional 0.999 g of catalyst was added.The mixture was placed under 50 PSI H₂ and was shaken at roomtemperature on the Parr shaker until the reaction was complete as judgedby reverse phase HPLC (5 days). The reaction mixture was filteredthrough celite to remove the catalyst. The celite was washed withmethanol. The filtrate was concentrated under reduced pressure on therotary evaporator. The crude product was dissolved in 200 ml ether andwashed with 100 ml H₂O and 50 ml H₂O. The organic layer was thenextracted with 2×50 ml portions of 1 M citric acid. The citric acidextract formed three layers. The bottom two layers were separated fromthe ether layer. Hexanes, 100 mL, were added to the ether layer and theorganic layer was extracted with an additional 50 mL of 1 M citric acid.The combined citric acid extracts were then extracted with 100 mLhexanes. The water extracts and the citric acid extracts were combinedand carefully neutralized to ˜pH 8.0 with Na₂CO₃. The basified solutionwas extracted with 2×200 mL ethyl acetate. The organic extracts weredried over Na₂SO₄, filtered and concentrated under reduced pressure onthe rotary evaporator. The crude product was mixed with 26.302 g ofglyoxylic acid, 25 mL diisopropylethylamine, 2.147 g 10% Pd/C, and 50 mLMeOH. The mixture was shaken under 50 PSI H₂ for two days. The reactionmixture was filtered through celite and then concentrated under reducedpressure on the rotary evaporator. The crude product was dissolved in200 mL ethyl acetate and extracted with 2×100 mL saturated NaHCO₃. Theorganic layer was washed with 100 mL 1 M NaH₂PO₄ and dried over Na₂SO₄.The reaction mixture was filtered and concentrated to provide 22.2 g ofthe crude product as a yellow oil. The crude product was purified bypouring the crude product onto a pad of flash silica % full in a 600 mLsintered glass funnel and eluting with a gradient of solvents. Thefunnel was eluted with 200 mL portions of 4×100% hexanes, 4×75:25hexanes/ethyl acetate, 4×1:1 hexanes/ethyl acetate, 4×25:75hexanes/ethyl acetate, 4×100% ethyl acetate, 3×100% CHCl₃, and 7×95:5CHCl₃/MeOH. The oily amber product, 17.648 g (45% yield) was found inthe 95:5 CHCl₃/MeOH fractions (3 to 7).

Four Step Method.

a. Synthesis of N-(2-((5-dibenzosuberyl)amino)ethyl)-1,2-ethanediamine,3. Dithethylenetriamine 1, 250 mL, was placed in a one liter three neckround bottom flask equipped with a magnetic stir bar. The solution wasplaced under an atmosphere of nitrogen and cooled in an ice bath to 4°C. The 12.108 g (5.29×10⁻² mol) of 5-chlorodibenzosuberane 2 was addedin spoonfuls over 10 min. The reaction was allowed to slowly warm toroom temperature and stir for 2.5 days. The reaction was then cooled inan ice bath and 350 mL of water was added. The solution was extractedwith 4×100 mL CH₂Cl₂. The organic layers were combined and washed with2×100 mL water. The organic layer was dried over sodium sulfate,filtered and concentrated on the rotary evaporator to afford 15.268 g(97.8% yield) of the crude product as an oil.

b. Synthesis ofN,N,N′,N″-Tetra((tert-butoxy-carbonyl)methyl)-N″-(2-((5-dibenzosuberyl)amino)ethyl)-1,2-ethanediamine5. The entire crude product from the previous reaction 3 was dissolvedin 75 mL of acetonitrile. Diisopropylethylamine, 68 mL was added to thereaction solution, which was flushed with nitrogen and cooled in an icebath. tert-Butyl bromoacetate 4, 40 mL (2.71×10⁻¹, 523 mol %) was addeddropwise to the reaction solution and the solution was allowed to slowlywarm to room temperature as it stirred overnight. The next day anadditional 7.5 mL of tert-butyl bromoacetate 4 was added to drive thereaction to completion. The reaction was stirred at room temperature forone more day and then concentrated under reduced pressure on the rotaryevaporator. Ethyl acetate, 200 mL was added and extraction was performedwith 3×100 mL of saturated sodium bicarbonate solution. The organiclayer was dried over sodium sulfate and concentrated under reducedpressure to obtain an amber oil. The oil was then further concentratedat 80° C. under hi-vacuum on the rotary evaporator to obtain 40.938 g ofthe crude product as an amber oil.

c. Synthesis ofN,N,N′,N″-Tetra((tert-butoxy-carbonyl)methyl)-N″-(2-(amino)ethyl)-1,2-ethanediamine.The crude product, 40.873 g, was dissolved in 40 mL methanol and placedin a 500 mL Parr hydrogenation bottle. Citric acid, 10.505 g was thenadded and the bottle was purged with nitrogen. The catalyst, 1.551 g of10% palladium on activated carbon, was then added to the Parr bottle.The mixture was then placed on the Parr shaker under 50 PSI H₂. Thehydrogenation was nearly complete after shaking for two days under 50PSI H₂ but the reaction was allowed to proceed for an additional twodays before it was removed from the Parr shaker. The reaction mixturewas filtered through celite. The filtrate was concentrated on the rotaryevaporator and then dissolved in 200 mL diethylether. The solution wascarefully mixed with 300 mL of a saturated NaHCO₃ solution. The etherlayer and the bicarbonate layers were separated. The bicarbonate layerwas back extracted with 2×50 mL of ether. The ether layers were combinedand mixed with 300 mL hexanes. The organic layer was then extracted with3×50 mL 1 M citric acid. The citric acid extracts were combined andextracted with 2×100 mL portions of 1:1 ether/hexanes solution (toremove traces of suberane). Sodium carbonate, 16.322 g was slowly addedto the citric acid solution with 100 mL of ethyl acetate on top of theaqueous layer. Additional sodium carbonate was added until the pH wasadjusted to pH 8 by pH paper. The solution was then extracted with 3×100mL ethyl acetate. The ethyl acetate extracts were combined and driedover sodium sulfate. The solution was filtered and concentrated on therotary evaporator to afford 21.755 g (75% yield) of the crude product asa yellow oil. The crude product was dissolved in 25 mL of ether andplaced on a pad of flash silica ¾ full in a 600 mL sintered glassfunnel. The funnel was eluted with 200 mL portions of 4×100% hexanes,4×9:1 hexanes/ethyl acetate, 4×75:25 hexanes/ethyl acetate, 8×50:50hexanes/ethyl acetate, 4×25:75 hexanes/ethyl acetate and 4×100% ethylacetate. The product appears to be present in all the fractions from75:25 hexanes/ethyl acetate to 100% ethyl acetate. The HPLC showsincreased levels of impurities in the 75:25 hexanes/ethyl acetatefractions as well as the first three 50:50 ethyl acetate/hexanesfractions. These fractions contain about 5.9 g of material. Theremaining fractions that contain product were combined to afford 11.332g (MH⁺ 560, 39% yield) of the oily product.

d. Synthesis of 1-tert-butoxy3,6,9-Tris((tert-butoxycarbonyl)methyl)-3,6,9-triazaundecanedioic Acid8. The purified product from the previous reaction 11.332 g wasdissolved in 40 mL methanol and placed under an atmosphere of nitrogenin a 500 mL Parr bottle. Glyoxylic acid monohydrate 6, 13.813 g wasadded to the solution followed by 1.102 g of 10% palladium on activatedcarbon. The bottle was placed on the Parr shaker under 50 PSI H₂. Themixture was shaken under overnight under H₂ and an aliquot tested thefollowing day revealed that the reaction was ˜90% complete. The reactionmixture was charged with 0.685 g of fresh catalyst and returned to thehydrogenator for three additional days at 50 PSI H₂. The reactionsolution was then filtered through celite, concentrated on the rotaryevaporator and dissolved in 200 mL ethyl acetate and dissolved in 200 mLethyl acetate. The ethyl acetate solution was carefully mixed with 175mL of saturated NaHCO₃ solution. The organic layer was washed with 100mL water followed by a wash with 100 mL 1M NaH₂PO₄ and finally 100 mLsaturated NaCl. The organic layer was dried over sodium sulfate,filtered and concentrated on the rotary evaporator to afford 10.444 g(84% yield) of a yellow oil.

Example 30 Activating and Conjugating DTPA

The DTPA, 5 g was dissolved in 40 mL 1.0 M tetrabutylammonium hydroxidein methanol. The methanol was removed under hi-vacuum to obtain aviscous oil. The oil was dissolved in 50 mL DMF and the volatilesolvents were removed under hi-vacuum on the rotary evaporator. The DMFtreatment was repeated two more times. The viscous oil was thendissolved in 50 ml DMF and mixed with 5 g HBTU. An 8 ml aliquot of theactivated DTPA solution was then added to the resin which was vortexmixed for 14 hr. The DTPA treatment was repeated until the resin gave anegative test for amines using the Kaiser test. Alternatively, DTPATetra-t-butyl ester could be used with conventional coupling agents suchas DIC and HBTU. (See Arano Y et al., J Med Chem. 1996 Aug. 30;39(18):3451-60).

Example 31 Conjugation of a Carboxylesterase to di-DTPA-Peptide

Carboxylesterase (5 mg) in 0.2 M phosphate buffer, pH 8.0, is treatedwith a five-fold molar excess of the cross-linking agentsulfo-succinimidyl-[4-maleimidomethyl]-cyclohexane-1-carboxylate(sulfo-SMCC). After stirring two hours at room temperature, theactivated enzyme is separated from low molecular weight contaminantsusing a spin-column of G-25 Sephadex and equilibrated in 0.1 M phosphatebuffer, pH 7, containing 1 mM EDTA. The tetrapeptideN-acetyl-Cys-Lys(DTPA)-Tyr-Lys(DTPA)-NH₂ (SEQ ID NO: 1) (ten-fold molarexcess) is added to the activated enzyme and dissolved in the samebuffer as used in the spin-column. After stirring for one hour at roomtemperature, the peptide carboxylesterase conjugate is purified fromunreacted peptide by spin-column chromatography on G-25 Sephadex in 0.25M acetate buffer, pH 6.0. Successful conjugation is demonstrated byindium-111 labeling of an aliquot of the conjugate, and analysis bysize-exclusion HPLC.

Example 32 Preparation of a Carboxylesterase-DTPA Conjugate

Two vials of rabbit liver carboxylesterase (SIGMA; protein content ˜17mg) are reconstituted in 2.2 ml of 0.1 M sodium phosphate buffer, pH 7.7and mixed with a 25-fold molar excess of CA-DTPA using a freshlyprepared stock solution (˜25 mg/ml) of the latter in DMSO. The finalconcentration of DMSO in the conjugation mixture is 3% (v/v). After 1hour of incubation, the mixture is pre-purified on two 5-mL spin-columns(Sephadex G50/80 in 0.1 M sodium phosphate pH 7.3) to remove excessreagent and DMSO. The eluate is purified on a TSK 3000G Supelco columnusing 0.2 M sodium phosphate pH 6.8 at 4 ml/min. The fraction containingconjugate is concentrated on a Centricon-10™ concentrator, andbuffer-exchanged with 0.1 M sodium acetate pH 6.5. Recovery: 0.9 ml,4.11 mg/ml (3.7 mg). Analytical HPLC analysis using standard conditions,with in-line UV detection, revealed a major peak with a retention timeof 9.3 min and a minor peak at 10.8 min in 95-to-5 ratio. Enzymaticanalysis showed 115 enzyme units/mg protein, comparable to unmodifiedcarboxylesterase. Mass spectral analyses (MALDI mode) of both unmodifiedand DTPA-modified CE shows an average DTPA substitution ratio near 1.5.A metal-binding assay using a known excess of indium spiked withradioactive indium confirmed the DTPA:enzyme ratio to be 1.24 and 1.41in duplicate experiments. Carboxylesterase-DTPA is labeled with In-111acetate at a specific activity of 12.0 mCi/mg, then treated with excessof non-radioactive indium acetate, and finally treated with 10 mM EDTAto scavenge off excess non-radioactive indium. Incorporation by HPLC andITLC analyses is 97.7%. A HPLC sample is completely complexed with a20-fold molar excess of bi-specific antibody hMN-14 Fab′×734 Fab′, andthe resultant product further complexes with WI2 (anti-ID to hMN-14),with the latter in 80-fold molar excess with respect to bi-specificantibody.

All patents and other references cited in the specification areindicative of the level of skill of those skilled in the art to whichthe invention pertains, and are incorporated by reference in theirentireties, including any tables and figures, to the same extent as ifeach reference had been incorporated by reference in its entiretyindividually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to obtain the ends and advantages mentioned,as well as those inherent therein. The methods, variances, andcompounds/compositions described herein as presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the invention.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention. Forexample, a variety of different binding pairs can be utilized, as wellas a variety of different therapeutic and diagnostic agents. Thus, suchadditional embodiments are within the scope of the present invention.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention.

1. A compound comprising one or more of the sequencesR¹-Lys(X)-R²-Lys(Y) or Lys(X)-R²-Lys(Y)-R¹, wherein (X) and (Y) arehaptens selected from the group consisting of DTPA and HSG, wherein R¹and R² are one or more amino acids, and wherein one or more effectormolecules are conjugated to R¹, R², (X), or (Y). 2-12. (canceled) 13.The compound of claim 1, wherein DTPA is attached to an indium cation.14-21. (canceled)
 22. The compound of claim 1, wherein the effectormolecule comprises one or more drugs, prodrugs, toxins, enzymes,oligonucleotides, radioisotopes, immunomodulators, cytokines, hormones,binding molecules, lipids, polymers, micelles, liposomes, nanoparticles,or combinations thereof.
 23. The compound of claim 22, wherein thebinding molecule comprises an antibody or a fragment thereof.
 24. Thecompound of claim 1, wherein the effector molecule comprises aplidin,azaribine, anastrozole, azacytidine, bleomycin, bortezomib,bryostatin-1, busulfan, calicheamycin, camptothecin,10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin,irinotecan (CPT-11), SN-38, carboplatin, cladribine, cyclophosphamide,cytarabine, dacarbazine, docetaxel, dactinomycin, daunomycinglucuronide, daunorubicin, dexamethasone, diethylstilbestrol,doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholinodoxorubicin, doxorubicin glucuronide, epirubicin glucuronide, ethinylestradiol, estramustine, etoposide, etoposide glucuronide, etoposidephosphate, floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO),fludarabine, flutamide, fluorouracil, fluoxymesterone, gemcitabine,hydroxyprogesterone caproate, hydroxyurea, idarubicin, ifosfamide,L-asparaginase, leucovorin, lomustine, mechlorethamine,medroprogesterone acetate, megestrol acetate, melphalan, mercaptopurine,6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin,mitotane, phenyl butyrate, prednisone, procarbazine, paclitaxel,pentostatin, PSI-341, semustine streptozocin, tamoxifen, taxanes, taxol,testosterone propionate, thalidomide, thioguanine, thiotepa, teniposide,topotecan, uracil mustard, velcade, vinblastine, vinorelbine,vincristine, ricin, abrin, ribonuclease, onconase, rapLR1, DNase I,Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtheria toxin, Pseudomonas exotoxin, Pseudomonas endotoxin, anantisense oligonucleotide, an interference RNA, or combinations thereof.25. The compound of claim 1, wherein the effector molecule comprisescamptothecin, doxorubicin, a camptothecin derivative or a doxorubicinderivative.
 26. (canceled)
 27. The compound of claim 1, furthercomprising an isotope selected from ¹⁸F, ³²P, ³³P, ⁴⁵Ti, ⁴⁷Sc, ⁵²Fe,⁵⁹Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁵Se, ⁷⁷As, ⁸⁶Y, ⁸⁹Sr, ⁸⁹Zr, ⁹⁰Y,⁹⁴Tc, ^(94m)Tc, ⁹⁹Mo, ^(99m)Tc, ¹⁰⁵Pd, ¹⁰⁵Rh, ¹¹¹Ag, ¹¹¹In, ¹²³I, ¹²⁴I,¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁵⁴⁻¹⁵⁸Gd, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho,¹⁶⁹Er, ¹⁷⁵Lu, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, ²¹¹At,²¹¹Pb ²¹²Bi, ²¹²Pb, ²¹³Bi, ²²³Ra, ²²⁵Ac, or combinations thereof. 28-32.(canceled)
 33. A method of treating and/or diagnosing a disease orcondition that may lead to a disease in a patient comprising: (A)administering to the patient a bispecific antibody that has at least onearm that binds a targeted tissue and at least one other arm that binds atargetable construct; (B) optionally, administering to the patient aclearing composition and allowing the composition to clear non-localizedbispecific antibodies from circulation; and (C) administering to thepatient a targetable construct comprising the compound of claim 1.34-47. (canceled)
 48. The method of claim 33, wherein the effectormolecule comprises one or more drugs, prodrugs, toxins, enzymes,radioisotopes, immunomodulators, oligonucleotides, cytokines, hormones,antibodies, or combinations thereof.
 49. The method of claim 33, whereinthe effector molecule comprises aplidin, azaribine, anastrozole,azacytidine, bleomycin, bortezomib, bryostatin-1, busulfan,camptothecin, 10-hydroxycamptothecin, carmustine, celebrex,chlorambucil, cisplatin, irinotecan (CPT-11), SN-38, carboplatin,cladribine, cyclophosphamide, cytarabine, dacarbazine, docetaxel,dactinomycin, daunomycin glucuronide, daunorubicin, dexamethasone,diethylstilbestrol, doxorubicin, 2-pyrrolinodoxorubicin (2P-DOX),cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicinglucuronide, ethinyl estradiol, estramustine, etoposide, etoposideglucuronide, etoposide phosphate, floxuridine (FUdR),3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, fluorouracil,fluoxymesterone, gemcitabine, hydroxyprogesterone caproate, hydroxyurea,idarubicin, ifosfamide, L-asparaginase, leucovorin, lomustine,mechlorethamine, medroprogesterone acetate, megestrol acetate,melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone,mithramycin, mitomycin, mitotane, phenyl butyrate, prednisone,procarbazine, paclitaxel, pentostatin, semustine streptozocin,tamoxifen, taxanes, taxol, testosterone propionate, thalidomide,thioguanine, thiotepa, teniposide, topotecan, uracil mustard,vinblastine, vinorelbine, vincristine, ricin, abrin, ribonuclease,onconase, rapLR1, DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin,Pseudomonas endotoxin, an antisense oligonucleotide, an interferenceRNA, or combinations thereof.
 50. The method of claim 33, wherein theeffector molecule comprises camptothecin, doxorubicin, a camptothecinderivative or a doxorubicin derivative.
 51. (canceled)
 52. The method ofclaim 33, wherein the compound further comprises an isotope selectedfrom ¹⁸F, ³²P, ³³P, ⁴⁵Ti, ⁴⁷Sc, ⁵²Fe, ⁵⁹Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga,⁶⁸Ga, ⁷⁵Se, ⁷⁷As, ⁸⁶Y, ⁸⁹Sr, ⁸⁹Zr, ⁹⁰Y, ⁹⁴Tc, ^(94m)Tc, ⁹⁹Mo, ^(99m)Tc,¹⁰⁵Pd, ¹⁰⁵Rh, ¹¹¹Ag, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm,¹⁵³Sm, ¹⁵⁴⁻¹⁵⁸Gd, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁶⁹Er, ¹⁷⁵Lu, ¹⁷⁷Lu, ¹⁸⁶Re,¹⁸⁸Re, ¹⁸⁹Re, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, ²¹¹At, ²¹¹Pb, ²¹²Bi, ²¹²Pb, ²¹³Bi,²²³Ra, ²²⁵Ac, or combination thereof.
 53. The method of claim 33,wherein the effector molecule comprises an enzyme selected from thegroup consisting of carboxylesterases, glucuronidases,carboxypeptidases, beta-lactamases, phosphatases, and mixtures thereof.54-58. (canceled)
 59. The method of claim 33, wherein the bispecificantibody comprises a human, chimeric or humanized antibody or a fragmentof a human, chimeric or humanized antibody.
 60. (canceled)
 61. Themethod of claim 33, wherein the bispecific antibody comprises a fusionprotein.
 62. (canceled)
 63. The method of claim 33, wherein the diseaseor condition comprises a malignant disease, a cardiovascular disease, aninfectious disease, an inflammatory disease, an autoimmune disease, ametabolic disease, or a neurological disease.
 64. The method of claim63, wherein the disease or condition comprises a malignant disease andthe targeted tissue comprises an antigen selected from the groupconsisting of carcinoembryonic antigen, tenascin, epidermal growthfactor receptor, platelet derived growth factor receptor, fibroblastgrowth factor receptors, vascular endothelial growth factor receptors,gangliosides, HER/2neu receptors and mixtures thereof.
 65. The method ofclaim 33, wherein the targeted tissue comprises a tumor.
 66. The methodof claim 65, wherein the tumor produces or is associated with antigensselected from the group consisting of colon-specific antigen-p (CSAp),carcinoembryonic antigen (CEA), CD4, CD5, CD8, CD14, CD15, CD19, CD20,CD21, CD22, CD23, CD25, CD30, CD45, CD74, CD80, HLA-DR, Ia, Ii, MUC 1,MUC 2, MUC 3, MUC 4, NCA, EGFR, HER 2/neu, PAM-4, TAG-72, EGP-1, EGP-2,A3, KS-1, Le(y), S100, PSMA, PSA, tenascin, folate receptor, VEGF, PIGF,ILGF-1, necrosis antigens, IL-2, IL-6, T101, MAGE, and combinationsthereof.
 67. The method of claim 33, wherein the targeted tissuecomprises a multiple myeloma, a B-cell malignancy, a T-cell malignancy,or combinations thereof.
 68. The method of claim 67, wherein the B-cellmalignancy is selected from the group consisting of indolent forms ofB-cell lymphomas, aggressive forms of B-cell lymphomas, chronicleukemias, multiple myeloma, and acute lymphatic leukemias.
 69. Themethod of claim 33, wherein the targeted tissue comprises a lymphomaincluding a non-Hodgkin's lymphoma or a Hodgkin's lymphoma. 70.(canceled)
 71. The method of claim 33, wherein the targeted tissuecomprises a melanoma, a carcinoma, a sarcoma, a glioma, or combinationsthereof.
 72. (canceled)
 73. The method of claim 63, wherein the diseaseor condition comprises a cardiovascular disease and the antibody orantibody fragment is specific for granulocytes, lymphocytes, monocytes,fibrin, D-dimer or a mixture thereof.
 74. (canceled)
 75. The method ofclaim 63, wherein the infectious disease is selected from the groupconsisting of abacterial disease, fungal disease, parasitic disease,viral disease, protozoan disease, mycoplasmal disease, and combinationsthereof.
 76. (canceled)
 77. The method of claim 63, wherein theautoimmune disease is selected from the group consisting of acuteidiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenicpurpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemiclupus erythematosus, lupus nephritis, rheumatic fever, polyglandularsyndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonleinpurpura, post-streptococcal-nephritis, erythema nodosum, Takayasu'sarteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis,sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy,polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome,thromboangitis obliterans, Sjogren's syndrome, primary biliarycirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronicactive hepatitis, polymyositis/dermatomyositis, polychondritis,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis, fibrosing alveolitis, and combinationsthereof.
 78. (canceled)
 79. The method of claim 33, further comprisingadministering one or more therapeutic or diagnostic agents.
 80. Themethod of claim 33, further comprising administering a therapeutic agentselected from antibodies, antibody fragments, drugs, prodrugs, toxins,enzymes, enzyme-inhibitors, nuclease, hormones, hormone antagonists,oligonucleotides, immunomodulators, cytokines, chelators, boroncompounds, uranium atoms, photoactive agents, radionuclides, andcombinations thereof.
 81. The method of claim 33, further comprisingadministering a cytokine selected from the group consisting of IL-1,IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, IL-21, interferon-α,interferon-β, interferon-γ, G-CSF, and GM-CSF, and mixtures thereof. 82.The method of claim 33, further comprising administering ananti-angiogenic agent selected from the group consisting of angiostatin,endostatin, baculostatin, canstatin, maspin, anti-VEGF antibodies,anti-placental growth factor antibodies, anti-vascular growth factorantibodies, and mixtures thereof.
 83. The method of claim 33, furthercomprising administering a diagnostic agent selected from radioisotopes,dyes, radioopaque materials, contrast agents, fluorescent compounds,enhancing agents, and combinations thereof.
 84. The method of claim 33,further comprising administering a metal selected from zinc, aluminum,gallium, lutetium, palladium, boron, gadolinium, uranium, manganese,iron, chromium, copper, cobalt, nickel, dysprosium, rhenium, europium,terbium, holmium, neodymium, and combinations thereof.
 85. (canceled)86. The method of claim 84, wherein the diagnostic agent comprises oneor more agents for photodynamic therapy. 87-88. (canceled)
 89. Themethod of claim 33, further comprising administering a therapeutic ordiagnostic nuclide selected from the group consisting of ¹⁸F, ³²P, ³³P,⁴⁵Ti, ⁴⁷Sc, ⁵²Fe, ⁵⁹Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁵Se, ⁷⁷As, ⁸⁶Y,⁸⁹Sr, ⁸⁹Zr, ⁹⁰Y, ⁹⁴Tc, ^(94m)Tc, ⁹⁹Mo, ^(99m)Tc, ¹⁰⁵Pd, ¹⁰⁵Rh, ¹¹¹Ag,¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁵⁴⁻¹⁵⁸Gd,¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁶⁹Er, ¹⁷⁵Lu, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ¹⁹⁴Ir,¹⁹⁸Au, ¹⁹⁹Au, ²¹¹At, ²¹¹Pb, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²²³Ra, ²²⁵Ac, andmixtures thereof.
 90. The method of claim 79, wherein the therapeuticagent comprises a therapeutic nuclide.
 91. The method of claim 90,wherein the therapeutic nuclide comprises ³²P, ³³P, ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu,⁶⁷Ga, ⁹⁰Y, ¹¹¹Ag, ¹¹¹In, ¹²³I, ¹³¹I, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho,¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹¹At, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²²³Ra, ²²⁵Ac, ormixtures thereof. 92-104. (canceled)
 105. The method of claim 33,further comprising performing an operative, intravascular, laparoscopic,or endoscopic procedure.
 106. The method of claim 33, wherein thebinding molecule is administered intravenously and the targetableconstruct is administered orally.